CN108739407A - A kind of underbrown japanese cherry tissue culture and rapid propagation method - Google Patents

Abstract

The invention discloses a kind of underbrown japanese cherry tissue culture and rapid propagation methods; this method has carried out sufficient experiment to each step of the tissue cultures of underbrown japanese cherry; the optimal culture condition of suitable underbrown japanese cherry tissue culture has been found out by experiment; establish underbrown japanese cherry tissue culture rapid propagation system; it overcomes the propagation methods such as traditional cuttage, grafting and needs a large amount of maternal plant; it also needs to spend a large amount of human and material resources, financial resources, time, and breeding coefficient is low, the slow problem of seedling.Its growth coefficient is big, rooting rate is good, and cultivation cycle is short, and survival rate is high.

Description

A kind of underbrown japanese cherry tissue culture and rapid propagation method

Technical field

The present invention relates to technical field of tissue culture, and in particular to a kind of underbrown japanese cherry tissue culture and rapid propagation method.

Background technology

Underbrown japanese cherry plant grace is beautiful, and blade is glossy, and flower is bright-coloured beautiful, is that seeds are spent in sight outstanding in afforestation. Be widely used in green way, cell, park, garden, river carry etc., afforestation effect is apparent.Traditional propagation method is mainly profit With propagation methods such as cuttage, graftings, but on the one hand these propagation methods need a large amount of maternal plant, on the other hand also need to spend a large amount of Human and material resources, financial resources, time etc., and breeding coefficient is low, seedling is slow.As oriental cherry is in city and small towns Urban Landscape Construction Extensive use, need large-scale nursery stock.Therefore, traditional propagation method far can not meet the needs in production.It is raw There is an urgent need to inquire into out the underbrown japanese cherry quick breeding technology that a kind of explant germination rate is high, growth coefficient is big, rooting rate is good in production, Push China's underbrown japanese cherry industrialized development.

With the development of modern biotechnology, people can in the environment of manual control using the totipotency of plant cell To be induced into plantlet using explants such as stem apex, stem section, the blades of plant, here it is plant tissue culture techniques.Plant group Training seedling has the features such as well developed root system, robust growth, resistance enhancing, extensive management, reduction production cost.Tissue culture technique is not Only have the advantages that breeding coefficient is big, the seedling period is short, and the stability of the excellent inhereditary feature of parent can be kept, is mountain cherry It spends quickly to breed and provides an effective approach with industrialized production.Also about the report of underbrown japanese cherry tissue culture technique at present It is fewer, and also system, some reports the present inventor also do not confirm that its growth coefficient is low by experiment, and rooting rate is low, relatively low Survival rate seriously constrain application of the underbrown japanese cherry in urban afforestation and gardens construction, therefore it is comprehensive urgently to establish a kind of system Underbrown japanese cherry tissue culturing system.

Invention content

The purpose of the present invention is to provide a kind of underbrown japanese cherry tissue culture and rapid propagation method, this method underbrown japanese cherry tissue cultures germination rates Height, growth coefficient is big, rooting rate is good, and cultivation cycle is short, and survival rate is high.

To achieve the above object, the technical scheme is that：

A kind of underbrown japanese cherry tissue culture and rapid propagation method, described method includes following steps：

(1) selection of explant：Growth selection is vigorous, and no disease and pests harm newly sends out the time short spray with suspend mode axillary bud Item is explant material；Crust green it is best, materials time in the sun-drenched morning it is best；It is light green to select epidermis as far as possible The significantly new branch of color suspend mode axillary bud form.The same day adopt under explant material on the day of must carry out disinfection processing, otherwise store Overlong time easily lead to axillary bud deriving failure；

(2) disinfection of explant；

(3) Primary culture：It will be cut into the segment of 2~3cm by the explant stem section disinfected, and ensures per segment On have 1 suspend mode axillary bud, with sterile razor blade first by the partially cut-away of stem section upper and lower side notch browning, rapidly by the bud of axillary bud stem section It is inoculated into primary culture medium upwards, is put in culturing room and cultivates after the completion of inoculation；The primary culture medium is：MS culture mediums+ 6-BA2~3mg/L+NAA 0.2~0.5mg/L+ sucrose 30g/L+ carragheens 8g/L；Condition of culture is：25 ± 2 DEG C of temperature, light It is 10h/d according to the time, intensity of illumination is 1500~2000Lx, humidity 60%-65%, pH value 5.8；

(4) Multiplying culture：First time Multiplying culture is continuing with primary culture medium, after cultivating 40d, the side that will newly sprout Bud tender stem is scaled off from explant stem section and is inoculated on proliferated culture medium；Starting the low algebraically period after proliferation, (the 2nd takes turns to the 5 wheels) select 1/2MS culture mediums or WPM culture mediums addition 6-BA 1~2mg/L, 0.05~0.2mg/L of NAA, sucrose 30g/L and Carragheen 8g/L；High algebraically Multiplying culture will be changed to 1~1.5mg/L of MS culture mediums addition 6-BA, NAA 0.1 (after the 6th wheel) ~0.2mg/L, sucrose 30g/L and carragheen 8g/L are best；Each stage condition of culture of Multiplying culture is unanimously：Condition of culture is： 25 ± 2 DEG C, light application time 12h/d of temperature, intensity of illumination are 2000~2500Lx, 60%~65%, pH 5.8~6.2；

(5) culture of rootage：The seedlings that later stage multiplicative stage is had reached to requirement of taking root (i.e. seedling plant height degree 3cm or more) are cut The callus of basal part of stem and invalid proliferation bud are removed, is inoculated on root media, the root media is：MS culture mediums+ IBA 0.2mg/L+NAA 0.2mg/L+AC 0.1mg/L+ sucrose 30g/L+ carragheens 8g/L；Condition of culture is：Temperature 28 ± 2 DEG C, light application time 8h/d, intensity of illumination is 1000~1500Lx, and humidity is 70%~80%, pH 6.0~6.2；

(6) hardening and transplanting.

Wherein it is preferred to which the disinfection specific method of explant is：The blade of shoot is cut along petiole base first, Long branch is cut into several sections with scissors, is placed in the water containing liquid detergent and cleans, and mostly vibrates back and forth several times, is careful not to make The crust of branch is damaged, is finally rinsed well under tap water, then branch is cut into stem section, every section of 2~3, band above with scissors Axillary bud is positioned in clean vial, is rinsed 1h with tap water flowing water, is outwelled the water in bottle, drain, cover bottle cap, take It is spare on aseptic operating platform.70% alcoholic solution, 2% liquor natrii hypochloritis, foot are got out on aseptic operating platform in advance The sterile water and other items of enough amounts opens the ultraviolet lamp disinfection 30 minutes or more in aseptic operating platform；

Then the explant cleaned up is cut into stem section with scissors, 2~3 axillary buds of every section of band above are positioned over clean Vial in, with tap water flowing water rinse 1h, outwell the water in bottle, drain, cover bottle cap, take standby on aseptic operating platform With；

Explant stem section is disinfected with 70% alcoholic solution, and processing time 15s outwells rapidly alcohol, then with 2% time Sodium chlorate solution handles 2min, outwells javelle water, is finally rinsed repeatedly with sterile water 4~5 times；

Furthermore it is preferred that hardening and the specific method of transplanting are：

The seedbed in greenhouse is selected to carry out hardening, warm indoor temperature is maintained at 20-25 DEG C, and humidity is maintained at 60%- 70%；The tissue-culture container seedling taken root is transferred on the seedbed in hardening greenhouse, 10d-15d is tamed under natural environment；3- before transplanting The bottle cap of tissue culture bottle is loosened, but do not opened completely by 4d；1-2d before transplanting, the bottle cap of tissue culture bottle is opened completely；Transplanting When, the rooted seedling by domestication is first lightly come out with tweezers from culture medium inner clip, by the culture medium clear water of root institute band It cleans up, is transplanted in hardening matrix completely, plastic covering film Small plastic shed keeps the environment of high temperature and humidity, induces new root Occur, the hardening time is 25-35d, after under growth root seedling grows new must shape root and restore normal growth, by the both ends of Small plastic shed It opens, slowly leaks informaton；Small plastic shed both ends are leaked informaton after 7d-10d, and Small plastic shed is opened completely, make it in greenhouse under natural conditions Growth.

It is further preferred that in above-mentioned acclimatization and transplants step, the hardening matrix is perlite, peat according to volume ratio 1: 4 ratio mixes.

Further, the multiplicative stage part lateral bud before oriental cherry seedling strain is taken root is not achieved directly into root media Standard or can not independent separated state of growing thickly, thus have to pass through strong seedling culture.By seedling plant height degree 2cm independent lateral buds below Or the Multiple Buds that several budlets connect together are inoculated into strong seedling culture base, budlet obviously grows up after culture 20~30 days, generally Reach into the standard taken root.Strong seedling culture base is that basic element of cell division dosage is greatly reduced on the basis of proliferated culture medium, suitably Auxin dosage is improved, minimal medium is that MS or WPM+ spends precious No.1,6-BA dosages 0.1~0.2mg/L, NAA dosage 0.1~ 0.2mg/L still adds sucrose 30g/L and carragheen 8g/L, and condition of culture is：25 ± 2 DEG C, light application time 12h/d of temperature, Intensity of illumination is 2000~2500Lx, 60%~65%, pH 5.8~6.2.

The invention has the advantages that：

The invention has carried out sufficient experiment to each step of the tissue cultures of underbrown japanese cherry, has been found out and has been suitble to by experiment The optimal culture condition of underbrown japanese cherry tissue culture establishes underbrown japanese cherry tissue culture rapid propagation system, overcomes the breedings such as traditional cuttage, grafting Method needs a large amount of maternal plant, also needs to spend a large amount of human and material resources, financial resources, time etc., and breeding coefficient is low, and seedling is slow to ask Topic.

This method underbrown japanese cherry tissue cultures germination rate is high, growth coefficient is big, rooting rate is good, and cultivation cycle is short, and survival rate It is high.

Description of the drawings

Fig. 1 is the shoot with suspend mode axillary bud；

Fig. 2 is hibernaculum pictorial diagram；

Fig. 3 is hibernaculum in startup stage pollution figure；

Fig. 4 is that the shoot with suspend mode axillary bud induces lateral bud in startup stage；

Fig. 5 is the low algebraically seedling strain of multiplicative stage；

Fig. 6 is the high algebraically seedling strain of multiplicative stage；

Fig. 7 is growing way situation in rooted seedling bottle.

Specific implementation mode

The following examples are used to illustrate the present invention, but are not intended to limit the scope of the present invention..Unless otherwise specified, of the invention All materials can be obtained by acquisition, purchase or complimentary manner.

In order to find the preferred technical solution of the present invention, applicant is logical to the various conditions for influencing underbrown japanese cherry tissue cultures Overtesting studies it, and to obtain the appropraite condition of suitable underbrown japanese cherry tissue-culturing rapid propagation, specific experiment is following (referring to Fig.1 ~Fig. 7)：

1, the selection of explant

Inventor selects the shoot (such as Fig. 1) with suspend mode axillary bud and hibernaculum (such as Fig. 2) to be tested respectively, as a result, Hibernaculum cannot continue, the shoot with suspend mode axillary bud does not have as explant in startup stage just all pollutions (such as Fig. 3) Pollution, and lateral bud (such as Fig. 4) can be induced.And directly gave up at that time without the spray of resting bud, in theory and practice Middle morning is proved unworkable.

Therefore (in late May, 2017) our growth selections are vigorous in testing, and no disease and pests harm, the new time short band that sends out are stopped The shoot of dormancy axillary bud is explant material；Crust green, the materials time is the sun-drenched morning；Selecting epidermis as far as possible is The significantly new branch of peak green suspend mode axillary bud form.The same day adopt under explant material on the day of must carry out disinfection processing, otherwise The overlong time of storage easilys lead to axillary bud deriving failure.

2, the pretreatment and disinfection of explant

The blade of shoot is cut along petiole base first, long branch is cut into several sections with scissors, be placed in containing It is cleaned in the water of liquid detergent, mostly vibrates the crust breakage for being careful not to make branch several times back and forth, finally rinsed under tap water dry Only, it then with scissors by branch is cut into stem section, 2~3 axillary buds of every section of band above are positioned in clean vial, use tap water Flowing water rinses 1h, outwells the water in bottle, drains, cover bottle cap, takes spare on aseptic operating platform.In advance on aseptic operating platform 70% alcoholic solution, 2% liquor natrii hypochloritis, sufficient amount of sterile water and other items are got out, aseptic operating platform is opened In ultraviolet lamp disinfection 30 minutes or more；

The selection of decontaminant concentration：Explant stem segments are disinfected with 70% alcoholic solution, processing time 15s, rapidly It outwells alcohol, then explant is handled respectively with the NaClO solution of 1%~4% various concentration of effective chlorine, it is different between 2~4min Time outwells thimerosal, is finally rinsed 4-5 times repeatedly with sterile water.It, will not during disinfecting with aseptic water washing It discontinuously shakes, to ensure that sterile water and thimerosal can be contacted with explant completely, improves disinfection efficiency.By tri- kinds of NaClO Effective chlorine density 1%, 2%, 4% and difference disinfecting time 2min, 3min, 4min are compared, it has been found that selection is effective Cl concn is 2%, and disinfecting time is best when being 2min, and living contaminants are minimum, injures minimum to explant, induction success rate is most Height, excessive concentration and overlong time can also kill oriental cherry explant simultaneously, reduce survival rate.

3, the selection of Primary culture condition

It will be cut into the segment of 2-3cm by the explant stem section disinfected, and ensures there is 1 suspend mode armpit on per segment The bud of axillary bud stem section is inoculated into opens rapidly by bud upwards with sterile razor blade first by the partially cut-away of stem section upper and lower side notch browning On dynamic culture medium, it is put in culturing room and cultivates after the completion of inoculation；All condition of culture are：25 ± 2 DEG C of temperature, light application time are 10h/d, intensity of illumination are 1500~2000Lx, humidity 60%-65%, pH value 5.8；

The selection of primary culture medium hormone kind and concentration：The axillary bud sprouting of induction explant dormant state is taken to go out lateral bud Mode, when culture devises addition sucrose 30g/L and carragheen 8g/L, pH value 5.8, then with regard to hormon in MS culture mediums And its proportioning carries out contrast test, it is specific to be respectively：

A1, MS+6-BA 1mg/L+NAA 0.1mg/L

A2, MS+6-BA 1mg/L+NAA 0.2mg/L+KT 0.2mg/L

A3, MS+6-BA 1mg/L+NAA 0.5mg/L+KT 0.5mg/L

A4, MS+6-BA 2mg/L+NAA 0.5mg/L

A5, MS+6-BA 2mg/L+NAA 0.1mg/L+KT 0.5mg/L

A6, MS+6-BA 2mg/L+NAA 0.2mg/L+KT 1mg/L；

A7, MS+6-BA 3mg/L+NAA 0.2mg/L

A8, MS+6-BA 3mg/L+NAA 0.5mg/L+KT 0.5mg/L

A9, MS+6-BA 3mg/L+NAA 0.1mg/L+KT 1mg/L

It is found by experiment that each combination has explant in varying numbers to sprout lateral bud, the apparent left and right of difference of sterilization method The height of induction success rate, under the premise of optimum sterilization method, 9 kinds of primary culture mediums have explant in varying numbers Body axillary bud sprouting goes out lateral bud, observes that the higher each combination explant stem section lower end callus volume of 6-BA concentration is bigger than normal, but It is that lateral bud growing way is vigorous, the light green area of blade is slightly larger, and three composite entity differences of a concentration of 1mg/L of 6-BA are little, have several Explant axillary bud sprouting is slightly partially slow, and lateral bud growing way is less than normal.The axillary bud sprouting respectively combined and lateral bud added with KT grow shape It is not notable that condition with the combination that do not add compares difference, it is contemplated that the requirement of production application, 6-BA concentration in 2~3mg/L, The primary culture medium of 0.2~0.5mg/L of NAA concentration is suitable for the explant Fiber differentiation of underbrown japanese cherry.

4, the selection of Multiplying culture stage difference proliferation algebraically period optimum medium

Corresponding primary culture medium before first time Multiplying culture is continued to use respectively, culture find proliferation seedling strain stem foot after 50 days There is different degrees of callus in portion, the higher hormone concentration the more apparent, and yellow green is generally presented in blade, there is slight Huang The trend of change, the MS class culture mediums that high hormone with high salt may be not suitable with proliferation seedling strain are related.The improvement direction of proliferated culture medium Being placed on reduces hormone and reduces in a great number of elements concentration, is not more than no more than 2mg/L and NAA dosages keeping 6-BA dosages Under the premise of 0.2mg/L, increase the culture medium of two kinds of less salts of 1/2MS and WPM, designs following 9 kinds of culture mediums, addition sucrose 30g/ L and carragheen 8g/L, each stage condition of culture of Multiplying culture are unanimously：25 ± 2 DEG C, light application time 12h/d of temperature, illumination is strong Degree is 2000-2500Lx, and 60%-65%, pH value 5.8 carries out contrast test to different types of culture medium, specific to be respectively：

B1, MS+6-BA 2mg/L+NAA 0.2mg/L

B2, MS+6-BA 1mg/L+NAA 0.1mg/L

B3, MS+6-BA 0.5mg/L+NAA 0.05mg/L

B4,1/2MS+6-BA 2mg/L+NAA 0.1mg/L

B5,1/2MS+6-BA 1mg/L+NAA 0.05mg/L

B6,1/2MS+6-BA 0.5mg/L+NAA 0.2mg/L

B7, WPM+6-BA 2mg/L+NAA 0.05mg/L

B8, WPM+6-BA 1mg/L+NAA 0.2mg/L

B9, WPM+6-BA 0.5mg/L+NAA 0.1mg/L

It is found afterwards three times by repeating experiment, under same hormonal readiness, in the 1/2MS culture mediums and WPM culture mediums of less salt Proliferation seedling strain it is more healthy and stronger than MS culture medium growing ways with high salt, the lateral bud form being proliferated out is apparent, and leaf color is dark green, and basal part of stem is cured Injured tissue is less, shortens to proliferating cycle 40 days.Therefore low algebraically period of the underbrown japanese cherry after starting proliferation, (the 2nd took turns to the 5th Wheel) select 0.05~0.2mg/L of 1/2MS culture mediums or WPM culture mediums addition 6-BA 1~2mg/L and NAA.

Proliferation proceeds to middle high algebraically (the 6th wheel) seedling strain later and further shortens within 30 days proliferating cycle, proliferation system Number is significantly raised, but is proliferated that lateral bud head is less than normal, and state of growing thickly is apparent, is proliferated in vain on the high side, and incubation time is more than after 30 days Proliferation seedling strain starts large quantities of yellows in bottle afterwards, it may be possible to which vigorous growth demand causes the nutriment in culture medium to consume in advance Totally, therefore subsequent proliferated culture medium needs to increase the content of mineral nutrition, attempts to train using MS culture mediums with high salt or WPM Foster base addition is a certain amount of to spend precious No.1 to solve the problems, such as this, specific to be respectively：

C1, MS+6-BA 0.5mg/L+NAA 0.05mg/L

C2, MS+6-BA 0.75mg/L+NAA 0.05mg/L

C3, MS+6-BA 1mg/L+NAA 0.05mg/L

C4, MS+6-BA 1mg/L+NAA 0.1mg/L

C5, MS+6-BA 1.5mg/L+NAA 0.2mg/L

C6, MS+6-BA 2mg/L+NAA 0.3mg/L

C7, WPM+6-BA 0.15mg/L+NAA 0.01mg/L+ spend precious No.1 0.5g/L

C8, WPM+6-BA 0.2mg/L+NAA 0.025mg/L+ spend precious No.1 1g/L

C9, WPM+6-BA 0.25mg/L+NAA 0.02mg/L+ spend precious No.1 0.5g/L

C10, WPM+6-BA 0.5mg/L+NAA 0.02mg/L+ spend precious No.1 1g/L

C11, WPM+6-BA0.5mg/L+NAA0.05mg/L+ spend precious No.1 0.5g/L

C12, WPM+6-BA 0.75mg/L+NAA 0.1mg/L+ spend precious No.1 1g/L

C13, WPM+6-BA 1mg/L+NAA0.1mg/L+ spend precious No.1 0.5g/L

C14, WPM+6-BA 1.5mg/L+NAA 0.25mg/L+ spend precious No.1 1g/L

By being found after experiment is repeated several times, growth coefficient decreases in each combination of the 6-BA concentration less than 1mg/L, seedling Strain blade growing way is excessively vigorous and stalk increment is little, and extension growth cycle effect is not also notable, is proliferated seedling strain difference in size Very big, whole growing way is irregular, is unfavorable for subsequent growth culture.Each group symphysis long period between 1~2mg/L of 6-BA concentration with It the increase of algebraically and gradually extends to 40 days or more, proliferation seedling strain size is integrally relatively uniform, and precious No.1 is spent in MS and WPM additions Culture medium in seedling strain growing way difference unobvious, due to spending precious No.1 higher price from the point of view of produce reality, MS classes Advantageously, from the point of view of comprehensive long-term cultivation situation, the high algebraically Multiplying culture of underbrown japanese cherry is with 6-BA in MS culture mediums by C3, C4, C5, C6 0.1~0.2mg/L of dosage 1~1.5mg/L, NAA dosage is best.

5, the selection of culture of rootage stage most suitable root media

The seedlings that later stage multiplicative stage is had reached to requirement of taking root (i.e. seedling plant height degree 3cm or more) cut being cured for basal part of stem Injured tissue and invalid proliferation bud, are inoculated on root media, every bottle is inoculated with 15 plants.For the most suitable root media of determination, with MS, WPM, 1/2MS be minimal medium, addition (0.2,0.5,1) mg/l IBA and (0.2,0.5,1.0) mg/l NAA and (0.1, 0.5,1) g/L activated carbons, add sucrose 30g/L and carragheen 8g/L, pH value 6.0, design the combination of following culture medium and carry out pair Than experiment：

D1, MS+IBA 0.2mg/L+NAA 0.2mg/L+AC 0.1mg/L

D2, MS+IBA 0.5mg/L+NAA 0.5mg/L+AC 0.5mg/L

D3, MS+IBA 1mg/L+NAA 1mg/L+AC 1mg/L

D4, WPM+IBA 0.2mg/L+NAA 0.5mg/L+AC 1mg/L

D5, WPM+IBA 0.5mg/L+NAA 0.2mg/L+AC0.1mg/L

D6, WPM+IBA 1mg/L+NAA 0.2mg/L+AC 0.5mg/L

D7,1/2MS+IBA 0.2mg/L+NAA 1mg/L+AC 0.5mg/L

D8,1/2MS+IBA0.5mg/L+NAA 0.2mg/L+AC 1mg/L

D9,1/2MS+IBA 1mg/L+NAA 0.5mg/L+AC 0.1mg/L

Be found by experiment that, IBA and NAA concentration be more than after 0.5mg/L rooted seedling strain basal part of stem callus clearly, Root is generally slightly long and grows on callus, very unfavorable for follow-up acclimatization and transplants.The higher root media of AC additive amounts Middle seedling strain ratio of taking root is obviously relatively low, in WPM and 1/2MS culture mediums seedling strain occur aetiolation successively after 30 days in culture, and Then unobvious in MS culture mediums.The root of seedling strain is elongated in D1, and callus is not notable, after 30 days the ratio of taking root can reach 70% with On, and seedling strain yellow is seldom, and seedling strain entirety growing way is preferable, therefore most suitable root media is D1, MS+IBA 0.2mg/L+ NAA 0.2mg/L+AC 0.1mg/L。

Embodiment

A kind of underbrown japanese cherry tissue culture and rapid propagation method, described method includes following steps：

(1) selection of explant：Growth selection is vigorous, and no disease and pests harm newly sends out the time short spray with suspend mode axillary bud Item is explant material；Crust green it is best, materials time in the sun-drenched morning it is best；It is light green to select epidermis as far as possible The significantly new branch of color suspend mode axillary bud form.The same day adopt under explant material on the day of must carry out disinfection processing, otherwise store Overlong time easily lead to axillary bud deriving failure；

(2) disinfection of explant：The blade of shoot is cut along petiole base first, long branch is cut into scissors It several sections, is placed in the water containing liquid detergent and cleans, mostly vibrate the crust breakage for being careful not to make branch several times back and forth, finally It is rinsed well under tap water, then branch is cut into stem section with scissors, 2~3 axillary buds of every section of band above are positioned over clean glass In glass bottle, 1h is rinsed with tap water flowing water, the water in bottle is outwelled, drains, cover bottle cap, take spare on aseptic operating platform.It carries It is preceding that the objects such as 70% alcoholic solution, 2% liquor natrii hypochloritis, sufficient amount of sterile water are got out on aseptic operating platform Product open the ultraviolet lamp disinfection 30 minutes or more in aseptic operating platform；

Then the explant cleaned up is cut into stem section with scissors, 2~3 axillary buds of every section of band above are positioned over clean Vial in, with tap water flowing water rinse 1h, outwell the water in bottle, drain, cover bottle cap, take standby on aseptic operating platform With；

Explant stem section is disinfected with 70% alcoholic solution, and processing time 15s outwells rapidly alcohol, then with 2% time Sodium chlorate solution handles 2min, outwells javelle water, is finally rinsed repeatedly with sterile water 4~5 times；

(3) Primary culture：It will be cut into the segment of 2~3cm by the explant stem section disinfected, and ensures per segment On have 1 suspend mode axillary bud, with sterile razor blade first by the partially cut-away of stem section upper and lower side notch browning, rapidly by the bud of axillary bud stem section It is inoculated into primary culture medium upwards, is put in culturing room and cultivates after the completion of inoculation；Condition of culture is：25 ± 2 DEG C of temperature, illumination Time is 10h/d, and intensity of illumination is 1500~2000Lx, humidity 60%-65%, pH value 5.8；

(4) Multiplying culture：First time Multiplying culture is continuing with primary culture medium, after cultivating 40d, the side that will newly sprout Bud tender stem is scaled off from explant stem section and is inoculated on proliferated culture medium；Starting the low algebraically period after proliferation, (the 2nd takes turns to the 5 wheels) select 1/2MS culture mediums or WPM culture mediums；High algebraically Multiplying culture will be changed to (after the 6th wheel) based on MS culture mediums Culture medium；Each stage condition of culture of Multiplying culture is unanimously：Condition of culture is：25 ± 2 DEG C, light application time 12h/d of temperature, light It is 2000~2500Lx, 60%~65%, pH5.8~6.2 according to intensity；

(5) culture of rootage：The seedlings that later stage multiplicative stage is had reached to requirement of taking root (i.e. seedling plant height degree 3cm or more) are cut The callus of basal part of stem and invalid proliferation bud are removed, is inoculated on root media；Condition of culture is：28 ± 2 DEG C of temperature, illumination Time is 8h/d, and intensity of illumination is 1000~1500Lx, and humidity is 70%~80%, pH 6.0~6.2；

(6) hardening and transplanting：The seedbed in greenhouse is selected to carry out hardening, warm indoor temperature is maintained at 20-25 DEG C, wet Degree is maintained at 60%-70%；The tissue-culture container seedling taken root is transferred on the seedbed in hardening greenhouse, 10d- is tamed under natural environment 15d；The bottle cap of tissue culture bottle is loosened, but do not opened completely by 3-4d before transplanting；1-2d before transplanting, the bottle cap of tissue culture bottle is complete It is complete to open；When transplanting, the rooted seedling by domestication is first lightly come out with tweezers from culture medium inner clip, by root institute band Culture medium is cleaned up completely with clear water, is transplanted in hardening matrix, and plastic covering film Small plastic shed keeps the ring of high temperature and humidity Border induces new root, and the hardening time is 25-35d, will after under growth root seedling grows new must shape root and restore normal growth The both ends of Small plastic shed are opened, and are slowly leaked informaton；Small plastic shed both ends are leaked informaton after 7d-10d, and Small plastic shed is opened completely, makes it in greenhouse It is grown under interior natural conditions.

According to the suitable culture medium in each stage that previous experiments obtain, its culture of each cultivation stage in specific each embodiment The selection of base and growth coefficient, rooting rate statistics such as table 1.

The culture medium in the different embodiment each stages of table 1 chooses situation

Therefore, from the point of view of the growth coefficient, rooting rate of embodiment 1-16, average coefficient of proliferation 5.4, average rooting rate It is 71%, growth coefficient is low during solving underbrown japanese cherry tissue culture, the low problem of rooting rate.

Although above having used general explanation and specific embodiment, the present invention is described in detail, at this On the basis of invention, it can be made some modifications or improvements, this will be apparent to those skilled in the art.Therefore, These modifications or improvements without departing from theon the basis of the spirit of the present invention belong to the scope of protection of present invention.

Claims (10)

1. a kind of underbrown japanese cherry tissue culture and rapid propagation method, which is characterized in that described method includes following steps：

(1) selection of explant：Growth selection is vigorous, and no disease and pests harm new send out the time short shoot with suspend mode axillary bud and is Explant material；

(2) explant is sterilized；

(3) Primary culture：It will be cut into the segment of 2-3cm by the explant stem section disinfected, and ensures there is 1 on per segment A suspend mode axillary bud, it is rapidly that the bud of axillary bud stem section is upward with sterile razor blade first by the partially cut-away of stem section upper and lower side notch browning It is inoculated into primary culture medium, is put in culturing room and cultivates after the completion of inoculation；The primary culture medium is：MS culture mediums+6-BA 2~3mg/L+NAA, 0.2~0.5mg/L+ sucrose 30g/L+ carragheens 8g/L；Condition of culture is：25 ± 2 DEG C of temperature, when illumination Between be 10h/d, intensity of illumination be 1500~2000Lx, humidity be 60%~65%, pH value 5.8；

(4) Multiplying culture：First time Multiplying culture is continuing with primary culture medium, after cultivating 40d, the lateral bud that will newly sprout Tender stem is scaled off from explant stem section and is inoculated on proliferated culture medium；The 2nd low algebraically for taking turns to the 5th wheel is proliferated after starting proliferation Cultivating the proliferated culture medium selected is：1/2MS culture medium+6-BA 1~2mg/L+NAA, 0.05~0.2mg/L+ sucrose 30g/L+ Carragheen 8g/L；The later high algebraically proliferated culture medium of 6th wheel will be changed to：1~1.5mg/L+NAA of MS culture medium+6-BA 0.1 ~0.2mg/L+ sucrose 30g/L+ carragheens 8g/L；Each stage condition of culture of Multiplying culture is：Condition of culture is：Temperature 25 ± 2 DEG C, light application time 12h/d, intensity of illumination is 2000~2500Lx, 60%~65%, pH 5.8~6.2；

(5) culture of rootage：Later stage multiplicative stage is had reached into requirement of taking root, i.e. the seedlings of seedling plant height degree 3cm or more cut stem The callus of base portion and invalid proliferation bud, are inoculated on root media, the root media is：MS culture mediums+IBA 0.2mg/L+NAA 0.2mg/L+AC 0.1mg/L+ sucrose 30g/L+ carragheens 8g/L；Condition of culture is：28 ± 2 DEG C of temperature, light It is 8h/d according to the time, intensity of illumination is 1000~1500Lx, and humidity is 70%~80%, pH 6.0~6.2；

(6) hardening and transplanting.

2. underbrown japanese cherry tissue culture and rapid propagation method according to claim 1, which is characterized in that explant selects the crust green to be Most preferably, it is best that the materials time, which is in the sun-drenched morning,.

3. underbrown japanese cherry tissue culture and rapid propagation method according to claim 1, which is characterized in that step (2) the explant disinfection Method be specially：Explant is cleaned first, the explant cleaned up is then cut into stem section, every section of band above with scissors 2~3 axillary buds, are positioned in clean vial, rinse 1h with tap water flowing water, outwell the water in bottle, drain, cover bottle Lid, takes spare on aseptic operating platform；

Explant stem section is disinfected with 70% alcoholic solution, and processing time 15s outwells rapidly alcohol, then with 2% hypochlorous acid Sodium solution handles 2min, outwells javelle water, is finally rinsed repeatedly with sterile water 4~5 times.

4. underbrown japanese cherry tissue culture and rapid propagation method according to claim 3, which is characterized in that using 70% alcoholic solution, It before 2% liquor natrii hypochloritis, sterile water, places it in aseptic operating platform first, opens the purple in aseptic operating platform Outer lamp sterilizes 30 minutes or more.

5. underbrown japanese cherry tissue culture and rapid propagation method according to claim 1, which is characterized in that low algebraically described in step (5) increases Growing the 1/2MS culture mediums in the proliferated culture medium that culture is selected can also use WPM culture mediums to substitute.

6. underbrown japanese cherry tissue culture and rapid propagation method according to claim 1, which is characterized in that the increasing before oriental cherry seedling strain is taken root Grow the stage, for part lateral bud be not achieved directly into culture of rootage standard or can not independent separated state of growing thickly, to first pass through Strong seedling culture, strong seedling culture base are：MS culture medium+6-BA 0.1~0.2mg/L+NAA, 0.1~0.2mg/L+ sucrose 30g/L+ Carragheen 8g/L, condition of culture are：25 ± 2 DEG C, light application time 12h/d of temperature, intensity of illumination are 2000~2500Lx, 60%-65%, pH 5.8~6.2.

7. underbrown japanese cherry tissue culture and rapid propagation method according to claim 6, which is characterized in that the MS culture mediums in strong seedling culture base It can also be substituted with the precious No.1 of WPM culture mediums+spend.

8. underbrown japanese cherry tissue culture and rapid propagation method according to claim 1, which is characterized in that hardening and the selection of time of transplanting are every The 3-7 months and the 9-11 months in year.

9. underbrown japanese cherry tissue culture and rapid propagation method according to claim 1, which is characterized in that the specific method of hardening and transplanting For：

The seedbed in greenhouse is selected to carry out hardening, warm indoor temperature is maintained at 20-25 DEG C, and humidity is maintained at 60%-70%； The tissue-culture container seedling taken root is transferred on the seedbed in hardening greenhouse, 10d-15d is tamed under natural environment；3-4d before transplanting, will The bottle cap of tissue culture bottle loosens, but not open completely；1-2d before transplanting, the bottle cap of tissue culture bottle is opened completely；It, will be through when transplanting The rooted seedling for crossing domestication, is first lightly come out from culture medium inner clip with tweezers, and the culture medium of root institute band is completely clear with clear water Wash clean is transplanted in hardening matrix, and plastic covering film Small plastic shed keeps the environment of high temperature and humidity, induces new root, refining The seedling time is 25-35d, and after under growth root seedling grows new must shape root and restore normal growth, the both ends of Small plastic shed are opened, are delayed Slow play wind；Small plastic shed both ends are leaked informaton after 7d-10d, and Small plastic shed is opened completely, it is made to be grown under natural conditions in greenhouse.

10. underbrown japanese cherry tissue culture and rapid propagation method according to claim 9, which is characterized in that the hardening matrix be perlite, Peat is mixed according to the ratio of volume ratio 1: 4.