CN107006367A - A kind of ' sunlight ' cherry tissue culture and rapid propagation method - Google Patents
A kind of ' sunlight ' cherry tissue culture and rapid propagation method Download PDFInfo
- Publication number
- CN107006367A CN107006367A CN201710259822.5A CN201710259822A CN107006367A CN 107006367 A CN107006367 A CN 107006367A CN 201710259822 A CN201710259822 A CN 201710259822A CN 107006367 A CN107006367 A CN 107006367A
- Authority
- CN
- China
- Prior art keywords
- culture
- sunlight
- seedling
- cherry
- rapid propagation
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
Classifications
-
- 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
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
- A01H4/00—Plant reproduction by tissue culture techniques ; Tissue culture techniques therefor
- A01H4/001—Culture apparatus for tissue culture
Abstract
The invention discloses a kind of tissue culture and rapid propagation method of ' sunlight ' cherry.The present invention is set by minimal medium, hormon composition and concentration level ratio optimization, semi-lignified branch is given birth to as explant using excellent strain of growing up then, through axillary bud deriving, shoot proliferation and culture of rootage, form intact plant, it is transplanted to after matrix, obtains the healthy and strong nursery stock that growth is neat, phenotype is consistent.The present invention has breeding coefficient high, and cultivation period is short, the advantages of not being subject to seasonal restrictions, and inductivity is up to 100%；Growth coefficient is up to 5.1, and the elongation growth of propagation bud is fast, and culture 18d buds are up to>2.5cm ,/bottle of effective bud average out to 35；Culture of rootage 10d, rooting rate>98%, radical is up to more than 5/plant；Tissue culture rooted seedling is healthy and strong, transplanting survival rate is up to more than 92%；Anniversary scale fast seedling growing can be carried out in production application, healthy and strong, ' sunlight ' cherry container seedling of neat and consistent is produced, has a extensive future.
Description
Technical field
The invention belongs to field of plant tissue culture technique, it is more particularly related to a kind of tissue culture of ' sunlight ' cherry
Quick-breeding method.
Background technology
' sunlight ' cherry (Cerasus youkou) is that day city lucky wild (C.yedoensis cv.Amagi-yoshin) is red with trembling with fear
The horticultural gardening kind of cherry (C.campanulata) cross breeding, is deciduous tree, tree-like cup-shaped；Hua Xianye is opened, umbrella shape flower
Sequence, 3 a branch of, and flower level is slightly sagging to carry out；The long mitriform of calyx tube, hairless, dark violet red or red, sepal oblong drapes over one's shoulders pin
Shape, full edge is hairless；Flower 3.8~4.6cm of footpath, spends 5 valves, and light red purple, vein is obvious, and tip deep 2 splits；Late March April at florescence
Just (middle and lower reach of Yangtze River), early and middle ten dayses in April (Beijing, Shandong).Its large flower and brilliant color, the florescence is lucky wild close with dye well, can be with dye Jing Ji
Open country collocation, forms the oriental cherry tunnel of red white phase contrast, and " cherry and wood " is referred to as in Japan, and extensive by people is liked.It is seen
Reward value is high, and tree vigo(u)r is strong, and disease-resistant resistance is strong, and heat resistance is that few in number at present can adapt to the southern and northern gas of China preferably
That waits views and admires Flowering Cherry Cultivars, and development prospect is wide.
At present, ' sunlight ' cherry is conventional is mainly bred using grafting, due to by fringe bar quantity, stock species, grafting
The limitation of the factors such as time, its reproductive number is limited, much can not meet the market demand.Conventional reproductive speed is slow, serious restriction
Its Rapid Popularization application, in the urgent need to carrying out expanding numerous by group culturation rapid propagating technology, is entered using the excellent strain of growing up of ' sunlight ' cherry as maternal plant
Row tissue culture scale breeding, promote ' sunlight ' cherry industrialization development, meaning reality and it is great.
By literature search, the report on ' sunlight ' cherry tissue-culturing rapid propagation is had not yet to see.If being planted with reference to cherry category is now delivered
Thing correlation plants tissue culture culture medium and carries out tissue-culturing rapid propagation to ' sunlight ' cherry, it may appear that tissue culture bud turns to be yellow, and elongation growth is slow, bud it is small and
It is weak, effective bud number is few, vitrification phenomenon situations such as, cause growth coefficient small, seedling is of poor quality, reproduction speed is slow, production cost
It is high.
The content of the invention
It is an object of the invention to：Overcome in the prior art on ' sunlight ' cherry tissue culture and rapid propagation method scarcity, related tissue culture
There is provided a kind of tissue culture and rapid propagation method of ' sunlight ' cherry the problem of poor to ' sunlight ' cherry tissue-culturing rapid propagation effect for culture medium.
In order to realize foregoing invention purpose, the invention provides a kind of ' sunlight ' cherry tissue culture and rapid propagation method, it includes as follows
Step：
(1) explant pretreatment and collection：Before clip explant, replaced every 3~4 days with carbendazim or Bravo solution
Spray ' sunlight ' cherry tree body 2~3 times, the upper semi-lignified branch of clip ' sunlight ' cherry is as explant (preferably fine
The morning 10 when or so clip), be placed under uviol lamp 20~30min of irradiation, disleaf is soaked after 5min with liquid detergent solution, made
Liquid detergent solution is dipped in cotton balls or banister bruss and carefully gently scrubs stem (being sure not to brush off the stipule of petiole base), then with pure
Water is cleaned, and low-temperature moisture preservation is standby；
(2) explant sterilization and axillary bud deriving：Explant is carried out disinfection using bromogeramine and mercuric chloride, is then seeded into
Culture obtains axillary bud on inducing culture, and inducing culture is using YG as minimal medium, and (6- benzyl amino glands are fast by every liter of addition 6-BA
Purine) 0.5~1.5mg, IBA (indolebutyric acid) 0~0.2mg, NAA (methyl α-naphthyl acetate) 0.05~0.2mg, 20~40g of sucrose, agar
6g, pH are 5.8~6.0；
(3) Multiplying culture：Axillary bud is cut to be inoculated on proliferated culture medium to cultivate and obtains breeding seedling, proliferated culture medium is with YG
For minimal medium, every liter of 0.4~1.0mg of addition 6-BA, 0.05~0.2mg of NAA, IBA0.05~0.2mg, GA3It is (red mould
Element) 0.5~1.5mg, 20~40g of sucrose, agar 6g, pH are 5.8~6.0；
(4) culture of rootage：The tender shoots for breeding seedling is cut and is inoculated into culture in root media and obtains rooted seedling, is taken root
Culture medium using 1/2YG culture mediums as minimal medium, every liter of addition NAA 0.2~0.5mg, IBA0.2~0.5mg, sucrose 15~
20g, agar 6g, pH are 5.8~6.0；
(5) hardening is with transplanting：Rooted seedling is moved on into greenhouse and carries out acclimatization and transplantses, ' sunlight ' cherry container seedling is obtained.
A kind of as ' sunlight ' cherry tissue culture and rapid propagation method of the invention improves, in step (2) and (3), the condition of culture
For 24 ± 1 DEG C, intensity of illumination 2000lx, light application time 12h/ days.
A kind of as ' sunlight ' cherry tissue culture and rapid propagation method of the invention improves, in step (4), and the condition of culture is 25 ±
1 DEG C, intensity of illumination 2500lx, light application time 12h/ days.
A kind of as ' sunlight ' cherry tissue culture and rapid propagation method of the invention improves, and the YG culture mediums contain following component：
1000mg/L NH4NO3、950mg/L KNO3、220mg/L CaCl2·2H2O、200mg/L Ca(NO3)2·4H2O、370mg/L
MgSO4·7H2O、300mg/L NaH2PO4、22.3mg/L MnSO4·4H2O、8.6mg/L ZnSO4·7H2O、6.2mg/L
H3BO3、0.83mg/L KI、0.25mg/L Na2MoO4·2H2O、0.25mg/L CuSO4·5H2O、0.025mg/L CoCl2、
33.4mg/L FeSO4·7H2O、44.8mg/L Na2·EDTA·H2O, 100mg/L inositol, 2.0mg/L glycine, 0.1mg/L
Thiamine hydrochloride, 0.5mg/L nicotinic acid, 0.5mg/L puridoxine hydrochlorides, pH 5.8, solvent is water；Described 1/2YG culture mediums are
By NH in YG culture mediums4NO3、KNO3、Ca(NO3)2·4H2O、CaCl2·2H2O、MgSO4·7H2O、NaH2PO4Consumption halve,
Remaining components unchanged.
A kind of as ' sunlight ' cherry tissue culture and rapid propagation method of the invention improves, and sterilization is to use 1wt% described in step (2)
Bromogeramine solution soak after 5~6min, outwell after bromogeramine solution with aseptic water washing 3~4 times, add
Soak after 1~5min, constantly shake therebetween in 0.1wt% mercuric chloride solutions, outwell and aseptic water washing is used after mercuric chloride solution 5~6 times.
A kind of as ' sunlight ' cherry tissue culture and rapid propagation method of the invention improves, and step is inoculated into Fiber differentiation described in (2)
It is to cut off explant sterile razor blade after stipule, petiole, the injury at stem section two ends on base, inoculates inducing culture
On, one bottle of inoculation, one explant.
A kind of as ' sunlight ' cherry tissue culture and rapid propagation method of the invention improves, and in step (3), axillary bud is cut and is inoculated into increasing
Grow after culture medium, axillary bud is differentiated to form sprouting clump, the bud height in sprouting clump is cut into 1.0~1.5cm's more than or equal to 3cm
Stem section, then culture in new proliferated culture medium is transferred to, obtain breeding seedling.
A kind of as ' sunlight ' cherry tissue culture and rapid propagation method of the invention improved, in step (5), by culture of rootage 10~15 days
Rooted seedling move on to environment 5~7 days adapted in greenhouse, finishing scouring seedling 1 day (by bottle cap from partly reaching standard-sized sheet) takes tissue-cultured seedling
Go out clean culture medium, be transplanted to volume ratio for peat：Perlite：Rice chaff ash=3:1:On 1 mixed-matrix, preceding 4~5 after transplanting
It uses epiphragma moisturizing, then unties epiphragma according to conventional seedling management.
The present invention passes through minimal medium (YG culture mediums, the basic culture set specifically designed for ' sunlight ' cherry physiological status
Base) setting, hormon composition and concentration level ratio optimization, semi-lignified branch is given birth to as explant using excellent strain of growing up then,
Through axillary bud deriving, shoot proliferation and culture of rootage, intact plant is formed, is transplanted to after matrix, obtain that growth is neat, phenotype is consistent
Healthy and strong nursery stock.Axillary bud deriving speed of the present invention with explant is fast, and inductivity is high, up to 100%；Breeding coefficient is high, reaches
5.1, cultivation period is short, the advantages of not being subject to seasonal restrictions, and the elongation growth of propagation bud is fast, and culture 18d buds are up to>3cm；Culture of rootage
10d, rooting rate>98%, radical is up to more than 5/plant；Tissue culture rooted seedling is healthy and strong, transplanting survival rate is up to more than 90%；In reality
Anniversary scale fast seedling growing can be carried out in production application, healthy and strong, ' sunlight ' cherry tissue-cultured seedling of neat and consistent is produced.
Relative to prior art, the invention has the advantages that：
The inventive method has that induced velocity is fast, inductivity is high, growth coefficient is big, clump bud is sturdy, elongation growth is fast, take root
Rate is high, well developed root system, and rooted seedling is sturdy, transplanting survival rate is high, and seedling growth is healthy and strong neat, the low advantage of production cost.This hair
Bright method provides effective way for the scale nursery stock production of ' sunlight ' cherry seedling, by scale breeding, can be extensive
Acquisition ' sunlight ' cherry seedling, by for ' sunlight ' cherry asxualization promote technical support is provided, be Guangdong or even South China garden
Woods is afforested and Ecological Civilization Construction provides new excellent Flowering Cherry Cultivars and high quality seedling, significant, is had a extensive future.
Brief description of the drawings
With reference to the accompanying drawings and detailed description, ' sunlight ' cherry tissue culture and rapid propagation method of the invention and beneficial effect are entered
Row is described in detail.
Fig. 1 is axillary bud deriving figure of the present invention.
Fig. 2 is the propagation bottle seedling in proliferated culture medium of the present invention.
Fig. 3 is the rooted seedling root system in root media of the present invention.
Fig. 4 is tissue culture rooted seedling of the present invention.
Fig. 5 is that tissue culture of the present invention is taken root the growing state after transplantation of seedlings.
Embodiment
In order that the purpose of the present invention, technical scheme and advantageous effects become apparent from, with reference to embodiments, to this
Invention is further elaborated.It should be appreciated that the embodiment described in this specification is just for the sake of this hair of explanation
It is bright, it is not intended to limit the present invention, parameter, the ratio of embodiment etc. can suit measures to local conditions to make a choice and have no substance to result
Influence.
YG culture mediums in following examples, contain following component：1000mg/L NH4NO3、950mg/L KNO3、
220mg/L CaCl2·2H2O、200mg/L Ca(NO3)2·4H2O、370mg/L MgSO4·7H2O、300mg/L NaH2PO4、
22.3mg/L MnSO4·4H2O、8.6mg/L ZnSO4·7H2O、6.2mg/L H3BO3、0.83mg/L KI、0.25mg/L
Na2MoO4·2H2O、0.25mg/L CuSO4·5H2O、0.025mg/L CoCl2、33.4mg/L FeSO4·7H2O、44.8mg/L
Na2·EDTA·H2O, 100mg/L inositol, 2.0mg/L glycine, 0.1mg/L thiamine hydrochlorides, 0.5mg/L nicotinic acid, 0.5mg/L
Puridoxine hydrochloride, solvent is water.1/2YG culture mediums are contained a great number of elements (NH during YG is cultivated4NO3、KNO3、Ca(NO3)2·
4H2O、CaCl2·2H2O、MgSO4·7H2O、NaH2PO4) consumption halve, remaining composition and preparation method are constant.
It is well mixed according to the composition and content of above-mentioned formula, adjusts pH 5.8,121 DEG C of 18~20min of sterilizing, obtain YG trainings
Base is supported, it is standby.
Embodiment 1
A kind of tissue culture and rapid propagation method of ' sunlight ' cherry, comprises the following steps：
(1) explant is gathered：During the fine morning 10 in 4~May or so, grown from clip on ' sunlight ' cherry maternal plant
The healthy and strong, semi-lignified branch of no disease and pests harm, laboratory is taken back after moisturizing processing, by spray disleaf after collection, clear with pure water
4 DEG C of refrigerators are put after wash clean as explant to save backup.Gather before explant, every 3~4d with 800 times of carbendazim, hundred bacterium
Clear alternately sprinkling sprouts branch 2~3 times.
(2) explant surface sterilization：Branch (explant) is cut into the long band axil stem sections of 4cm or so, in ultra-clean work
First scrubbed, then gone out solution with the new gill of mass fraction 1% with sterilized water on platform, 5~6min of soak time outwells new gill
Go out after solution, with aseptic water washing 3 times, then add the mercuric chloride solution of mass fraction 0.1%, soaked according to the tender degree of explant children
1~5min, constantly shakes, outwells after mercuric chloride solution therebetween, then with aseptic water washing 5~6 times.Aseptic filter paper blots sterilizable material
Surface water drops, are cut off after stipule, petiole, stem section two ends injury with sterile razor blade, then stem section is cut into 2cm band axil stem sections
Band axil cut-out that is standby, being sterilized.
(3) axillary bud deriving：The band axil stem section (explant) disinfected is inoculated on inducing culture and cultivated, bar is cultivated
Part is：24 ± 1 DEG C, intensity of illumination 2000lx, light application time 12h/d, one explant of every bottle of inoculation, axillary bud 3d starts to sprout
Dynamic, axillary bud elongation is fast, and 18d can be grown to 2.5~3cm (Fig. 1), and inductivity is 100%, and inducing culture is using YG as basic culture
Base, (hormone is purchased from Sigma-Aldrich by every liter of addition 6-BA 0.8mg, IBA0.1mg, NAA0.1mg, sucrose 30g, agar 6g
Company), pH is that 5.8 (its compound method is：After mentioned component is well mixed, pH value is adjusted, is sterilized standby), thus obtain armpit
Bud.
(4) Multiplying culture：The axillary bud induced is cut into 1.0~1.5cm stem section to transfer culture on proliferated culture medium,
Condition of culture is：24 ± 1 DEG C, intensity of illumination 2000lx, light application time 12h/d, proliferated culture medium is using YG as minimal medium, often
Rise addition 6-BA 0.6mg, IBA 0.1mg, NAA 0.1mg, GA30.5mg, sucrose 30g, agar 6g, pH are 5.8 (its preparation side
Method is：After mentioned component is well mixed, pH value is adjusted, is sterilized standby), axillary bud is accessed after proliferated culture medium, and bud elongation growth is fast,
Simple bud formation bud clump, the trend (table 1) that growth coefficient subtracts afterwards with the extension of Subculture Time in first increasing, 18d growth coefficients highest,
Up to 5.1, height of seedling>2.5cm ,/bottle of effective bud average out to 35 (Fig. 2) thus obtains breeding seedling.
The influence that the different subculture cycles of table 1 are bred to ' sunlight ' cherry
(5) culture of rootage：Choosing healthy and strong effective bud from propagation seedling, (bud is high>1.5cm) cut and be transferred to root media
Middle culture, condition of culture is：25 ± 1 DEG C, intensity of illumination 2500lx, light application time 12h/d, root media is using 1/2YG as base
Basal culture medium, every liter of addition NAA 0.2mg, IBA 0.5mg, sucrose 15g, agar 6g, pH is 5.8 (its compound method is：Will be upper
State composition it is well mixed after, adjust pH value, sterilize standby), 10d rooting rates>98%, every plant averagely take root number up to more than 5 (Fig. 3,
Fig. 4).
(6) hardening is with transplanting：The bottle seedling that will take root moves on in greenhouse adaptation 5~7d of external environment, then by bottle cap from
The seedling in bottle is carefully taken out after partly reaching standard-sized sheet hardening 1d, hardening, cleans and sticks the culture medium on seedling, be transplanted to and fill up mud
Charcoal:Perlite:Rice chaff ash=3:1:In the seedling-raising cup of 1 (volume ratio) mixed-matrix, each glass of one young plant of plantation, transplanting earthing is deep
Degree just covers root system, compresses matrix, is in close contact seedling root and matrix, root water of being drenched after the completion of transplanting.
(7) seedling culture：After transplanting, the management of moisture, fertilizer and anti-bacteria is carried out to seedling：
1. water management：Matrix keeps moistening to bottle seedling after the transfer, and relative air humidity is advisable 80%~90%, and preceding 4
~5d can control 25 ± 1 DEG C of cultivation temperature by epiphragma moisturizing, afterwards by moisturizing of spraying, and cool canopy is hidden with 70% sunshade net
Light；
2. the management of fertilizer and anti-bacteria：Sprayed after the transfer with the Bravo of mass fraction 0.1% or carbendazim solution within the 3rd day
Mist sterilization, afterwards every 10d sprayings once, Bravo and carbendazim are used alternatingly；When having, sprouting is sprouted, new root is grown
When, 1500 times of nutrient solutions are sprayed, according to seedling growth, are sprayed once every 15d；
3. stabilization to be grown, grows after sprouting and new root, is stepped up intensity of illumination, is routinely managed until carrying out full exposure
Reason, transplants latter month survival rate and is higher than 96% (Fig. 5), when seedling length is high to 15cm (' sunlight ' cherry seedling), can be moved to big
Field cultivating large seedling.
Embodiment 2
A kind of tissue culture and rapid propagation method of ' sunlight ' cherry, comprises the following steps：
(1) explant is gathered：During the fine morning 10 in 4~May or so, grown from clip on ' sunlight ' cherry maternal plant
The healthy and strong, semi-lignified branch of no disease and pests harm, laboratory is taken back after moisturizing processing, by spray disleaf after collection, clear with pure water
4 DEG C of refrigerators are put after wash clean as explant to save backup.Gather before explant, every 3~4d with 800 times of carbendazim, hundred bacterium
Clear alternately sprinkling sprouts branch 2~3 times.
(2) explant surface sterilization：Branch (explant) is cut into the long band axil stem sections of 4cm or so, in ultra-clean work
First scrubbed, then gone out solution with the new gill of mass fraction 1% with sterilized water on platform, 5~6min of soak time outwells new gill
Go out after solution, with aseptic water washing 3 times, then add the mercuric chloride solution of mass fraction 0.1%, soaked according to the tender degree of explant children
1~5min, constantly shakes, outwells after mercuric chloride solution therebetween, then with aseptic water washing 5~6 times.Aseptic filter paper blots sterilizable material
Surface water drops, are cut off after stipule, petiole, stem section two ends injury with sterile razor blade, then stem section is cut into 2cm band axil stem sections
Band axil cut-out that is standby, being sterilized.
(3) axillary bud deriving：The band axil stem section (explant) disinfected is inoculated on inducing culture and cultivated, bar is cultivated
Part is：24 ± 1 DEG C, intensity of illumination 2000lx, light application time 12h/d, one explant of every bottle of inoculation, axillary bud 3d starts to sprout
Dynamic, bud growing way is good, and inductivity is 90.7%, and (table 2 is visible, and YG is ' sunlight ' cherry armpit by minimal medium of YG for inducing culture
Bud induces optimal minimal medium), every liter of addition 6-BA1.0mg, NAA0.1mg, sucrose 30g, (hormone is purchased from agar 6g
Sigma-Aldrich companies), pH is that 5.8 (its compound method is：After mentioned component is well mixed, pH value is adjusted, is sterilized standby
With), thus obtain axillary bud.
Inducing effect of ' sunlight ' cherry of table 2 on different minimal mediums
(4) Multiplying culture：The axillary bud induced is cut into 1.0~1.5cm stem section to transfer culture on proliferated culture medium,
Condition of culture is：24 ± 1 DEG C, intensity of illumination 2000lx, light application time 12h/d, proliferated culture medium is using YG as minimal medium, often
Rise addition 6-BA 0.4mg, IBA 0.1mg, NAA 0.05mg, GA31.0mg, sucrose 30g, agar 6g, pH are 5.8 (its preparation
Method is：After mentioned component is well mixed, pH value is adjusted, is sterilized standby), axillary bud is accessed after proliferated culture medium, 18d growth coefficients
Up to 3.2, leaf green, unfold, stem is relatively strengthened, and internode length is general, thus obtains breeding seedling.
(5) culture of rootage：Choosing healthy and strong effective bud from propagation seedling, (bud is high>1.5cm) cut and be transferred to root media
Middle culture, condition of culture is：25 ± 1 DEG C, intensity of illumination 2500lx, light application time 12h/d, described root media is with 1/
2YG is minimal medium, and every liter of addition NAA 0.2mg, IBA 0.2mg, sucrose 15g, agar 6g, pH are 5.8 (its compound method
For：By mentioned component it is well mixed after, adjust pH value, sterilize standby), 10d rooting rates up to 83.7%,.
(6) hardening is with transplanting：The bottle seedling that will take root moves on in greenhouse adaptation 5~7d of external environment, then by bottle cap from
The seedling in bottle is carefully taken out after partly reaching standard-sized sheet hardening 1d, hardening, cleans and sticks the culture medium on seedling, be transplanted to and fill up mud
Charcoal:Perlite:Rice chaff ash=3:1:In the seedling-raising cup of 1 (volume ratio) mixed-matrix, each glass of one young plant of plantation, transplanting earthing is deep
Degree just covers root system, compresses matrix, is in close contact seedling root and matrix, root water of being drenched after the completion of transplanting.
(7) seedling culture：After transplanting, the management of moisture, fertilizer and anti-bacteria is carried out to seedling：
1. water management：Matrix keeps moistening to bottle seedling after the transfer, and relative air humidity is advisable 80%~90%, and preceding 4
~5d can control 25 ± 1 DEG C of cultivation temperature by epiphragma moisturizing, afterwards by moisturizing of spraying, and cool canopy is hidden with 70% sunshade net
Light；
2. the management of fertilizer and anti-bacteria：Sprayed after the transfer with the Bravo of mass fraction 0.1% or carbendazim solution within the 3rd day
Mist sterilization, afterwards every 10d sprayings once, Bravo and carbendazim are used alternatingly；When having, sprouting is sprouted, new root is grown
When, 1500 times of nutrient solutions are sprayed, according to seedling growth, are sprayed once every 15d；
3. stabilization to be grown, grows after sprouting and new root, is stepped up intensity of illumination, is routinely managed until carrying out full exposure
Reason, transplants latter month survival rate and is higher than 93%, when seedling length is high to 15cm (' sunlight ' cherry seedling), can be moved to crop field cultivation
Seedlings.
Embodiment 3
A kind of tissue culture and rapid propagation method of ' sunlight ' cherry, comprises the following steps：
(1) explant is gathered：During the fine morning 10 in 4~May or so, grown from clip on ' sunlight ' cherry maternal plant
The healthy and strong, semi-lignified branch of no disease and pests harm, laboratory is taken back after moisturizing processing, by spray disleaf after collection, clear with pure water
4 DEG C of refrigerators are put after wash clean as explant to save backup.Gather before explant, every 3~4d with 800 times of carbendazim, hundred bacterium
Clear alternately sprinkling sprouts branch 2~3 times.
(2) explant surface sterilization：Branch (explant) is cut into the long band axil stem sections of 4cm or so, in ultra-clean work
First scrubbed, then gone out solution with the new gill of mass fraction 1% with sterilized water on platform, 5~6min of soak time outwells new gill
Go out after solution, with aseptic water washing 3 times, then add the mercuric chloride solution of mass fraction 0.1%, soaked according to the tender degree of explant children
1~5min, constantly shakes, outwells after mercuric chloride solution therebetween, then with aseptic water washing 5~6 times.Aseptic filter paper blots sterilizable material
Surface water drops, are cut off after stipule, petiole, stem section two ends injury with sterile razor blade, then stem section is cut into 2cm band axil stem sections
Band axil cut-out that is standby, being sterilized.
(3) axillary bud deriving：The band axil stem section (explant) disinfected is inoculated on inducing culture and cultivated, bar is cultivated
Part is：24 ± 1 DEG C, intensity of illumination 2000lx, light application time 12h/d, one explant of every bottle of inoculation, axillary bud 3d starts to sprout
Dynamic, bud growing way is good, and inductivity is 89%, described inducing culture using YG as minimal medium, every liter of addition 6-BA 1.0mg,
IBA 0.1mg, NAA 0.1mg sucrose 30g, agar 6g (hormone is purchased from Sigma-Aldrich companies), pH is 5.8 (its preparation side
Method is：After mentioned component is well mixed, pH value is adjusted, is sterilized standby), thus obtain axillary bud.
(4) Multiplying culture：The axillary bud induced is cut into 1.0~1.5cm stem section to transfer culture on proliferated culture medium,
Condition of culture is：24 ± 1 DEG C, intensity of illumination 2000lx, light application time 12h/d, proliferated culture medium is using YG as minimal medium, often
Rise addition 6-BA 0.8mg, IBA 0.2mg, NAA 0.05mg, GA31.0mg, sucrose 35g, agar 6g, pH are 5.8 (its preparation
Method is：After mentioned component is well mixed, pH value is adjusted, is sterilized standby), axillary bud is accessed after proliferated culture medium, 18d growth coefficients
Up to 4.3, leaf green, unfold, stem is slightly general, and internode length is general, thus obtains breeding seedling.
(5) culture of rootage：Choosing healthy and strong effective bud from propagation seedling, (bud is high>1.5cm) cut and be transferred to root media
Middle culture, condition of culture is：25 ± 1 DEG C, intensity of illumination 2500lx, light application time 12h/d, described root media is with 1/
2YG is minimal medium, and every liter of addition NAA 0.5mg, IBA0.2mg, sucrose 20g, agar 6g, pH are 5.8 (its compound method
For：By mentioned component it is well mixed after, adjust pH value, sterilize standby), 10d rooting rates up to 91%,.
(6) hardening is with transplanting：The bottle seedling that will take root moves on in greenhouse adaptation 5~7d of external environment, then by bottle cap from
The seedling in bottle is carefully taken out after partly reaching standard-sized sheet hardening 1d, hardening, cleans and sticks the culture medium on seedling, be transplanted to and fill up mud
Charcoal:Perlite:Rice chaff ash=3:1:In the seedling-raising cup of 1 (volume ratio) mixed-matrix, each glass of one young plant of plantation, transplanting earthing is deep
Degree just covers root system, compresses matrix, is in close contact seedling root and matrix, root water of being drenched after the completion of transplanting.
(7) seedling culture：After transplanting, the management of moisture, fertilizer and anti-bacteria is carried out to seedling：
1. water management：Matrix keeps moistening to bottle seedling after the transfer, and relative air humidity is advisable 80%~90%, and preceding 4
~5d can control 25 ± 1 DEG C of cultivation temperature by epiphragma moisturizing, afterwards by moisturizing of spraying, and cool canopy is hidden with 70% sunshade net
Light；
2. the management of fertilizer and anti-bacteria：Sprayed after the transfer with the Bravo of mass fraction 0.1% or carbendazim solution within the 3rd day
Mist sterilization, afterwards every 10d sprayings once, Bravo and carbendazim are used alternatingly；When having, sprouting is sprouted, new root is grown
When, 1500 times of nutrient solutions are sprayed, according to seedling growth, are sprayed once every 15d；
3. stabilization to be grown, grows after sprouting and new root, is stepped up intensity of illumination, is routinely managed until carrying out full exposure
Reason, transplants latter month survival rate and is higher than 92%, when seedling length is high to 15cm (' sunlight ' cherry seedling), can be moved to crop field cultivation
Seedlings.
The announcement and teaching of book according to the above description, those skilled in the art in the invention can also be to above-mentioned embodiment party
Formula carries out appropriate change and modification.Therefore, the invention is not limited in embodiment disclosed and described above, to this
Some modifications and changes of invention should also be as falling into the scope of the claims of the present invention.Although in addition, this specification
In used some specific terms, but these terms are merely for convenience of description, do not constitute any limitation to the present invention.
Claims (9)
1. a kind of ' sunlight ' cherry tissue culture and rapid propagation method, it is characterised in that comprise the following steps：
(1) explant pretreatment and collection：Before clip explant, alternately sprayed with carbendazim or Bravo solution every 3~4 days
' sunlight ' cherry tree body 2~3 times, the upper semi-lignified branch of clip ' sunlight ' cherry is placed under uviol lamp as explant and irradiates 20
~30min, disleaf is soaked with liquid detergent solution and cleaned after 5min, low-temperature moisture preservation is standby；
(2) explant sterilization and axillary bud deriving：Explant is carried out disinfection using bromogeramine and mercuric chloride, induction is then seeded into
Culture obtains axillary bud on culture medium, and inducing culture is using YG as minimal medium, every liter of 0.5~1.5mg of addition 6-BA, IBA0
~0.2mg, 0.05~0.2mg of NAA, 20~40g of sucrose, agar 6g, pH are 5.8~6.0；
(3) Multiplying culture：Axillary bud is cut to be inoculated on proliferated culture medium to cultivate and obtains breeding seedling, proliferated culture medium is using YG as base
Basal culture medium, every liter of 0.4~1.0mg of addition 6-BA, 0.05~0.2mg of NAA, IBA0.05~0.2mg, GA30.5~
1.5mg, 20~40g of sucrose, agar 6g, pH are 5.8~6.0；
(4) culture of rootage：The tender shoots for breeding seedling is cut and is inoculated into culture in root media and obtains rooted seedling, culture of rootage
Base is using 1/2YG culture mediums as minimal medium, every liter of 0.2~0.5mg of addition NAA, IBA0.2~0.5mg, 15~20g of sucrose,
Agar 6g, pH are 5.8~6.0；
(5) hardening is with transplanting：Rooted seedling is moved on into greenhouse and carries out acclimatization and transplantses, ' sunlight ' cherry container seedling is obtained.
2. ' sunlight ' cherry tissue culture and rapid propagation method according to claim 1, it is characterised in that in step (2), the culture bar
Part is 24 ± 1 DEG C, intensity of illumination 2000lx, light application time 12h/ days.
3. ' sunlight ' cherry tissue culture and rapid propagation method according to claim 1, it is characterised in that in step (3), the culture bar
Part is 24 ± 1 DEG C, intensity of illumination 2000lx, light application time 12h/ days.
4. ' sunlight ' cherry tissue culture and rapid propagation method according to claim 1, it is characterised in that in step (4), the culture bar
Part is 25 ± 1 DEG C, intensity of illumination 2500lx, light application time 12h/ days.
5. ' sunlight ' cherry tissue culture and rapid propagation method according to claim 1, it is characterised in that the YG culture mediums contain following
Composition：1000mg/L NH4NO3、950mg/L KNO3、220mg/L CaCl2·2H2O、200mg/L Ca(NO3)2·4H2O、
370mg/L MgSO4·7H2O、300mg/L NaH2PO4、22.3mg/L MnSO4·4H2O、8.6mg/L ZnSO4·7H2O、
6.2mg/L H3BO3、0.83mg/L KI、0.25mg/L Na2MoO4·2H2O、0.25mg/L CuSO4·5H2O、0.025mg/L
CoCl2、33.4mg/L FeSO4·7H2O、44.8mg/L Na2·EDTA·H2O, 100mg/L inositol, 2.0mg/L glycine,
0.1mg/L thiamine hydrochlorides, 0.5mg/L nicotinic acid, 0.5mg/L puridoxine hydrochlorides, pH 5.8, solvent is water；Described 1/2YG trainings
It is by NH in YG culture mediums to support base4NO3、KNO3、Ca(NO3)2·4H2O、CaCl2·2H2O、MgSO4·7H2O、NaH2PO4Use
Amount halves, remaining components unchanged.
6. ' sunlight ' cherry tissue culture and rapid propagation method according to claim 1, it is characterised in that being sterilized described in step (2) is
Soaked using 1wt% bromogeramine solution after 5~6min, with aseptic water washing 3~4 times, add 0.1wt% mercuric chloride solutions
After 1~5min of middle immersion, with aseptic water washing 5~6 times.
7. ' sunlight ' cherry tissue culture and rapid propagation method according to claim 1, it is characterised in that step is inoculated into described in (2)
It is to cut off explant sterile razor blade after stipule, petiole, the injury at stem section two ends on inducing culture, inoculates and lure
Lead on culture medium.
8. ' sunlight ' cherry tissue culture and rapid propagation method according to claim 1, it is characterised in that in step (3), axillary bud is cut
It is inoculated into after proliferated culture medium, axillary bud is differentiated to form sprouting clump, the bud height in sprouting clump is cut into 1.0 more than or equal to 3cm
~1.5cm stem section, then culture in new proliferated culture medium is transferred to, obtain breeding seedling.
9. ' sunlight ' cherry tissue culture and rapid propagation method according to claim 1, it is characterised in that in step (5), rooted seedling is moved
Environment is adapted into greenhouse 5~7 days, tissue-cultured seedling is removed and cleans culture medium by finishing scouring seedling 1 day, is transplanted to volume ratio for peat：It is precious
Zhu Yan：Rice chaff ash=3:1:On 1 mixed-matrix, use epiphragma moisturizing within first 4~5 days after transplanting, then untie epiphragma according to normal
Advise seedling management.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710259822.5A CN107006367B (en) | 2017-04-20 | 2017-04-20 | 'sunshine' cherry tissue culture rapid propagation method |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710259822.5A CN107006367B (en) | 2017-04-20 | 2017-04-20 | 'sunshine' cherry tissue culture rapid propagation method |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN107006367A true CN107006367A (en) | 2017-08-04 |
| CN107006367B CN107006367B (en) | 2019-12-13 |
Family
ID=59446821
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201710259822.5A Active CN107006367B (en) | 2017-04-20 | 2017-04-20 | 'sunshine' cherry tissue culture rapid propagation method |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN107006367B (en) |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110476813A (en) * | 2019-09-03 | 2019-11-22 | 江苏农林职业技术学院 | A kind of oriental cherry rapid propagation in vitro method |
| CN114403003A (en) * | 2022-01-12 | 2022-04-29 | 中国科学院合肥物质科学研究院 | In-vitro regeneration method of green cherry plant |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP3801871B2 (en) * | 2001-02-26 | 2006-07-26 | 独立行政法人科学技術振興機構 | Method for inducing adventitious buds of sweet cherry cultivars by plant tissue culture |
| CN105340742A (en) * | 2015-11-16 | 2016-02-24 | 广州天适集团有限公司 | Tissue culture rapid propagation method for cerasus yunnanensis(Franch.)Yu et Li adult excellent single plant 'Guangzhou' cerasus yunnanensis |
| CN105379621A (en) * | 2015-11-16 | 2016-03-09 | 华南农业大学 | Efficient in-vitro plant regeneration method of adult high-quality single-plant Xiaoqiao oriental cherry of cerasus lannesiana var. speciosa |
| CN106538392A (en) * | 2016-12-08 | 2017-03-29 | 上海杉植物科技有限公司 | A kind of white oriental cherry tissue culture and rapid proliferation method |
-
2017
- 2017-04-20 CN CN201710259822.5A patent/CN107006367B/en active Active
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP3801871B2 (en) * | 2001-02-26 | 2006-07-26 | 独立行政法人科学技術振興機構 | Method for inducing adventitious buds of sweet cherry cultivars by plant tissue culture |
| CN105340742A (en) * | 2015-11-16 | 2016-02-24 | 广州天适集团有限公司 | Tissue culture rapid propagation method for cerasus yunnanensis(Franch.)Yu et Li adult excellent single plant 'Guangzhou' cerasus yunnanensis |
| CN105379621A (en) * | 2015-11-16 | 2016-03-09 | 华南农业大学 | Efficient in-vitro plant regeneration method of adult high-quality single-plant Xiaoqiao oriental cherry of cerasus lannesiana var. speciosa |
| CN106538392A (en) * | 2016-12-08 | 2017-03-29 | 上海杉植物科技有限公司 | A kind of white oriental cherry tissue culture and rapid proliferation method |
Non-Patent Citations (1)
| Title |
|---|
| 叶小玲等: "云南樱桃优良单株的组织培养与快速繁殖", 《植物生理学报》 * |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110476813A (en) * | 2019-09-03 | 2019-11-22 | 江苏农林职业技术学院 | A kind of oriental cherry rapid propagation in vitro method |
| CN110476813B (en) * | 2019-09-03 | 2021-11-30 | 江苏农林职业技术学院 | In-vitro rapid propagation method for oriental cherry |
| CN114403003A (en) * | 2022-01-12 | 2022-04-29 | 中国科学院合肥物质科学研究院 | In-vitro regeneration method of green cherry plant |
| CN114403003B (en) * | 2022-01-12 | 2022-11-04 | 中国科学院合肥物质科学研究院 | In-vitro regeneration method for green cherry blossom plants |
Also Published As
| Publication number | Publication date |
|---|---|
| CN107006367B (en) | 2019-12-13 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN101849506A (en) | Tissue culture and rapid propagation method of acer palmatum | |
| CN106577281B (en) | The high planting percent breeding method of polygala arillata stem section tissue culture | |
| CN105613287B (en) | A kind of method bucket cotton rose tissue rapid propagation method for culturing seedlings | |
| CN109618927A (en) | A kind of method for tissue culture of sweetautumn clematis | |
| CN109392715A (en) | A kind of tissue culture and rapid propagation method of Kiwi berry gold and jade | |
| CN103430842A (en) | Quick propagation method of hybrid orchid tissue culture | |
| CN103430854A (en) | Tissue culturing method of clematis guernsey cream | |
| CN109717075A (en) | A method of hemerocailis middendorffi regeneration plant is obtained by evoking adventive bud | |
| CN107197746A (en) | A kind of mating system of China fir field excellent resources | |
| CN107047305A (en) | A kind of quick breeding method for tissue culture of dendrobium seedling | |
| CN107006367A (en) | A kind of ' sunlight ' cherry tissue culture and rapid propagation method | |
| CN105379621B (en) | A kind of high-efficiency in-vitro plant regeneration method of Prunus donarium adult fine individual plant " little Qiao " cherry | |
| CN108782247A (en) | A kind of method for tissue culture of late cherry " Yu Yihuang " kind of Japan | |
| CN105123526B (en) | Germplasm storage method of zinnia elegan tissue culture propagation | |
| CN105340742B (en) | A kind of tissue culture and rapid propagation method of Yunnan cherry adult fine individual plant " Guangzhou " cherry | |
| CN104604735B (en) | A kind of tissue culture and rapid propagation method of U.S.'s red autumnal leaves Lagerstroemia indica L. | |
| CN106665354A (en) | Rapid propagation method of potted gerbera jamesonii bolus tissue culture | |
| CN108077066A (en) | A kind of caladium bicolor somatic embryogenesis pathway tissue culture and rapid propagation method | |
| CN107135943A (en) | A kind of winter cherry rapid propagation in vitro method | |
| CN109287484A (en) | Dwarf form bigleaf hydrangea method for tissue culture | |
| CN109729976A (en) | A kind of floral leaf fernleaf hedge bamboo tissue culture and rapid propagation method | |
| CN109392714A (en) | A kind of tissue culture and rapid propagation method of Kiwi berry gold plum | |
| CN105766643B (en) | Method of the explant to improve Dangshan pear tissue culture shoot survival percent is obtained based on cutting back branch in 8-9 months | |
| CN107182783A (en) | A kind of method of Hainan rhizomes of Panax notoginseng bastem portion regeneration induction plant | |
| CN108782244B (en) | Tissue culture method for longzhuguo |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |