CN105379621B - A kind of high-efficiency in-vitro plant regeneration method of Prunus donarium adult fine individual plant " little Qiao " cherry - Google Patents
A kind of high-efficiency in-vitro plant regeneration method of Prunus donarium adult fine individual plant " little Qiao " cherry Download PDFInfo
- Publication number
- CN105379621B CN105379621B CN201510786966.7A CN201510786966A CN105379621B CN 105379621 B CN105379621 B CN 105379621B CN 201510786966 A CN201510786966 A CN 201510786966A CN 105379621 B CN105379621 B CN 105379621B
- Authority
- CN
- China
- Prior art keywords
- culture
- seedling
- cherry
- explant
- plant
- 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.)
- Active
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
- A01H4/005—Methods for micropropagation; Vegetative plant propagation using cell or tissue culture techniques
-
- 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 high-efficiency in-vitro plant regeneration method of Prunus donarium adult fine individual plant ' little Qiao ' cherry.The present invention is set by minimal medium, hormon composition and concentration level ratio optimization, young sprout is given birth to as explant using excellent strain of growing up then, through axillary bud deriving, shoot proliferation and culture of rootage, forms intact plant, after being transplanted to matrix, the healthy and strong nursery stock that growth is neat, phenotype is consistent is obtained.The present invention has the axillary bud speed of explant fast；Inductivity is high, up to 100%；Growth coefficient is big, up to more than 4.5, and propagation bud elongation growth is fast, and culture 15d buds are up to 3~4cm；Rooting rate is up to 100%, every plant of mean elements more than 5；Tissue culture rooted seedling is healthy and strong, transplanting survival rate is high.Anniversary scale fast seedling growing can be carried out in production application, healthy and strong, ' little Qiao ' cherry seedling of neat and consistent is produced, has a extensive future.
Description
Technical field：
The invention belongs to the technical field of tissue culture of plant, and in particular to a kind of Prunus donarium adult fine individual plant ' little Qiao '
The high-efficiency in-vitro plant regeneration method of cherry.
Background technology：
Prunus donarium (Cerasus lannesiana var.speciosa) is subordinate to the rose family (Rosaceae) cherry category
(Cerasus) Japan, is originated in, is mainly distributed on her the beans peninsula.Prunus donarium is both good ornamental plant, is also conventional foodstuff.
Its blade has special aromatic odor, is the traditional food material that Japanese likes, supply falls short of demand in market, has great expectations of and passes through import
Cherry leaf meets domestic markets demand.In view of the economic value of Prunus donarium, institute of woods section of Foshan city and Dujiangyan City are successively from day
This introducing and planting, it is processed into semi-finished product for ornamental plantation and receipts leaf and exports Japan back.Due to introducing a fine variety environmental condition, breeding and cultivation
The influence of the factors such as technology, its plant scale is govern always.' little Qiao ' cherry is Guangzhou Tian Shi Group Co., Ltd from Prunus donarium
In select suitable In Guangdong Province plantation excellent fancy breed, its spring bud green, bud white to pink, petal it is white
Color, edge or with blush, 3.0~3.8cm of Hua Jing；Fragrance is spent, 2~5 a branch of；Late Febuary at florescence is to mid or late March.It is ' small
It is tall ' cherry likes subacidity soil, Xi Shui, manurial-philous, happiness sunlight, but avoids ponding, and optimum growth temperature is 15 DEG C~28 DEG C, wide
State, Shenzhen, can normally it get over the summer from change, Dongguan, Qingyuan City, Shaoguan and other places, growth is normal, and it is vigorous that the flowers are in blossom, turns into South China's sight
The important kind of oriental cherry is appreciated, DEVELOPMENT PROSPECT is wide.
' little Qiao ' cherry is the excellent fancy breed selected from Prunus donarium, and its propagation method refers to the numerous of Prunus donarium
Grow mode.Prunus donarium Sterile culture uses seed seedling-raising, but because Prunus donarium originates in Japan, setting percentage is low, percentage of seedgermination is low,
Only 30% or so.Its seedlings differentiation of growing directly from seeds is big, unstable, to ensure the good characteristic of ' little Qiao ' cherry, should not pass through seed
Breeding.It can be bred in Prunus donarium production by cuttage, cutting medium, cuttings source, degree of lignification, HORMONE TREATMENT are dense
Degree etc. is very big on cuttage survival rate influence, wherein inserting epicormic branch cuttage survival rate is much higher than cuttage survival rate, such as takes group
Train seedling spray carry out cuttage rooting rate (>79%) it is higher than the rooting rate (47.8%) of seedling cutting cuttage.' little Qiao ' cherry is numerous
It is slow that the problem of cuttage survival rate is extremely low, reproductive speed are equally existed during educating, seriously governs its Rapid Popularization.With ' little Qiao '
Cherry carries out tissue culture scale breeding to be maternal, realizes that breeding asxualization is promoted, and is the important channel of ' little Qiao ' cherry industrialization development.
The tissue culture technique report of Prunus donarium is seldom, only two at present (tissue cultures of Prunus donarium and breeding,
1994, Zhou Zhijian etc.；The industrial breeding technique of Prunus donarium, 1996, Liu Songsong etc.), the group of this two report Prunus donariums
Training technology is identical, and the main distinction is that female parent material used is different, and the former female parent material used is 2 plants of seedlings that grow directly from seeds, Hou Zhesuo
With the Prunus donarium seedling (the annual tissue-cultured seedling that fringe bar comes out from the former tissue culture) that female parent material is cutting propagation.Ground more than
Study carefully report in it is visible, Prunus donarium tissue culture technique still has the following disadvantages：1. the female parent material of tissue culture for grow directly from seeds seedling or
Person's cutting propagation seedling, it is not the adult select tree by Long-term selection；2. subculture cycle is grown, 25~30d；3. the bud of squamous subculture
Small, only 5~10mm is high, it is impossible to is directly used in culture of rootage.4. process of rooting culture is complicated, efficiency is low, needs first by subculture budlet
Strong bud 10~15d of culture is carried out, treats that bud can grow to 2~3cm, then is transferred in root media and carries out culture of rootage, strong sprout rate is low,
Only 60% subculture bud can extend；5. the decline of subculture bud is fast, after subculture 15~16 times, proliferation rate reduces, under subculture bud quality
Drop, need to re-replace material.6. carrying out tissue-culturing rapid propagation using the technology, speed is slow, and cost is high, it is difficult to realizes scale.7. by
Different from the physiological status that adult is set greatly in treelet, it is big that the tissue culture scheme using treelet as female parent material not necessarily can be suitably used for adult
Tree, this all exists on many seeds.Empirical tests, it is wooden that the select tree that grown up using the tissue culture scheme to ' little Qiao ' gives birth to half then
Change spray and carry out tissue cultures, the bud elongation of Multiplying culture is slow, of poor quality, propagation multiplying power is low, and vitrification phenomenon is serious, rooting rate
Low, technology is unstable.
The content of the invention：
The purpose of the present invention be for grow up present in the existing tissue culture technique of ' little Qiao ' cherry select tree tissue culture propagation without
Fungus strain is established difficult, and the bud elongation of Multiplying culture is slow, of poor quality, vitrification phenomenon is serious, subculture cycle length, culture of rootage mistake
Journey is complicated, rooting rate is low, the technical problems such as technology is unstable, and provides a kind of height of Prunus donarium adult fine individual plant ' little Qiao ' cherry
In vitro plant regeneration method is imitated, this method has fast adventitious bud inducing, propagation multiplying power height, the sturdy elongation of bud is fast, rooting rate is high, raw
The features such as offspring well developed root system is healthy and strong, transplanting survival rate.
The present invention is grown up using Prunus donarium, and raw semi-lignified spray is explant to fine individual plant ' little Qiao ' cherry then, by basic
Culture medium design, the optimization of hormone kind, concentration and its proportioning, its in vitro plant regeneration technique efficiently, stable is established, is reached
Breed the mesh that bud stalwartness elongation growth is fast, subculture cycle is short, proliferation rate is high, rooting rate is high, rooted seedling is healthy and strong, transplanting survival rate is high
's.Asxualization popularization is carried out by scale breeding, will be high-quality oriental cherry needed for Guangdong or even South China's afforestation, beautification
The breeding of seedling provides technical support, and economic benefit is huge.
The high-efficiency in-vitro plant regeneration method of Prunus donarium adult fine individual plant ' little Qiao ' cherry of the present invention, it is characterised in that
Comprise the following steps：
A, axillary bud deriving：It is outer that the conduct of semi-lignified young sprout is given birth to then in spring, on clip ' little Qiao ' cherry adult select tree maternal plant
Implant, culture on inducing culture is inoculated into after sterilized and obtains axillary bud, condition of culture is：25 ± 2 DEG C, the μ of intensity of illumination 60
mol.m-2.s-1, light application time 12h/d, described inducing culture is that every liter contains 1.0~2.0mg of 6-BA, NAA 0.05
~0.2mg, 25~40g of sucrose, the agar of constant, surplus are XMD culture mediums, and pH is 5.8~6.0；
B, Multiplying culture：Axillary bud is cut to be inoculated on proliferated culture medium to cultivate and obtains breeding seedling, condition of culture is：25±
2 DEG C, 60 μm of ol.m of intensity of illumination-2.s-1, light application time 12h/d, described proliferated culture medium is, every liter containing 6-BA 0.5~
0.05~0.1mg of 1.5mg, NAA, 25~40g of sucrose, constant agar, surplus are XMD culture mediums, and pH is 5.8~6.0；
C, culture of rootage：Tender stem is cut from propagation seedling it is inoculated into root media culture and obtain rooted seedling, cultivates bar
Part is：25 ± 2 DEG C, 60 μm of ol.m of intensity of illumination-2.s-1, light application time 12h/d, described root media is that every liter contains
0.5~1.5mg of NAA, 15~20g of sucrose, constant agar, surplus are XMD culture mediums, and pH is 5.8~6.0；
Rooted seedling is moved on into hardening in greenhouse, then takes out seedling, cleans and sticks the culture medium on seedling, be transplanted to by volume
Compare peat soil：Yellow mud：Vermiculite=2:1:In 1 mixed-matrix, training orientation is carried out, obtains ' little Qiao ' cherry seedling；
Described XMD culture mediums, every liter contains NH4NO3800mg、KNO3400mg、CaCl2·2H2O 200mg、Ca
(NO3)2·2H2O 200mg、MgSO4·7H2O 370mg、KH2PO4170mg、MnSO4·4H2O 22.3mg、ZnSO4·7H2O
8.6mg、H3BO39.3mg、KI 0.83mg、Na2MoO4·2H2O 0.25mg、CuSO4·5H2O 0.05mg、CoCl20.025mg、
FeSO4·7H2O 27.8mg、Na2·EDTA·H2O 37.3mg、Myo-Inositol 100mg、Glycine2mg、
ThiamineHCl 0.1mg, Nicotinicacid 0.5mg, PyridoxineHCl 0.5mg, surplus are water, pH
5.8.The XMD culture mediums be according to the big tree physiological status of ' little Qiao ' cherry adult, cultured in vitro nutritional need and the XMD bases that design
Basal culture medium.
Semi-lignified young sprout is given birth on described clip ' little Qiao ' cherry maternal plant then as explant preferably in clip explant
Before, replace ' little Qiao ' cherry adult select tree maternal plant tree body 4~5 times selected by sprinkling every 10d carbendazim, Bravo, then clip
Semi-lignified young sprout is given birth on ' little Qiao ' cherry maternal plant then as explant, disleaf, is cleaned up by the use of pure water as explant, it is low
Warm moisturizing is standby.
Described sterilization is preferably：Explant is soaked into 7~8min with the bromogeramine solution of mass fraction 0.5%~1%,
After outwelling bromogeramine solution, 20 are soaked with aseptic water washing 3~4 times, then with the alcohol water blend of volume fraction 70~75%
~30s, after outwelling alcohol water blend, add in the mercuric chloride solution of mass fraction 0.05%~0.1%, according to the tender degree of explant children
1~5min is soaked, constantly shakes therebetween, outwells mercuric chloride solution, then with aseptic water washing 5~6 times, the explant after being sterilized
Body.
It is inoculated into after described sterilization on inducing culture and the explant after sterilization is preferably cut off into leaf with sterile razor blade
After handle, stem section both ends injury, inoculate on inducing culture.
Described Multiplying culture preferably cuts axillary bud, is inoculated on proliferated culture medium and cultivates, axillary bud is differentiated to form newly
Bud clump, the stem section that the edible tender branch of bud height >=3cm in sprouting clump is cut into 1.0~1.5cm turn to be linked into new propagation again
Culture obtains breeding seedling in culture medium, and condition of culture is：25 ± 2 DEG C, 60 μm of ol.m of intensity of illumination-2.s-1, light application time 12h/
D, described proliferated culture medium are that every liter contains 0.5~1.5mg of 6-BA, 0.05~0.1mg of NAA, 25~40g of sucrose, constant
Agar, surplus are XMD culture mediums, and pH is 5.8~6.0.
The present invention by minimal medium (XMD culture mediums, specifically for according to the big tree physiological status of ' little Qiao ' cherry adult,
The nutritional need of cultured in vitro and design) setting, hormon composition and concentration level ratio optimization, to grow up excellent strain then
Raw semi-lignified young sprout is explant, through axillary bud deriving, shoot proliferation and culture of rootage, forms intact plant, is transplanted to matrix
Afterwards, the healthy and strong nursery stock that growth is neat, phenotype is consistent is obtained.The present invention has the axillary bud speed of explant fast；Inductivity is high, reaches
100%；Growth coefficient is big, up to more than 4.5, and propagation bud elongation growth is fast, and culture 15d buds are up to 3~4cm；Rooting rate is reachable
100%, every plant of mean elements more than 5；Tissue culture rooted seedling is healthy and strong, transplanting survival rate is high.It can be carried out in production application
Anniversary scale fast seedling growing, healthy and strong, ' little Qiao ' cherry seedling of neat and consistent is produced, is had a extensive future.
The present invention is had the following advantages and effect relative to prior art：Induced velocity is fast, inductivity is high；Propagation system
Number is big, clump bud is sturdy, elongation growth is fast；Rooting rate is high, and well developed root system, rooted seedling is sturdy, transplanting survival rate is high, and seedling growth is good for
It is strong neat.By scale breeding, it will be promoted for the asxualization of ' little Qiao ' cherry and technical support is provided, and be Guangdong or even South China garden
Woods greening supplies high quality seedling guarantee, there is extremely important meaning.
Brief description of the drawings：
Fig. 1 and Fig. 2 is the adventitious bud bud induction of select tree sterilizable material；
Fig. 3 is the propagation bud in proliferated culture medium；
Fig. 4 is the bud high measurement for breeding bud；
Fig. 5 is the rooted seedling root system in root media；
Fig. 6 is tissue culture rooted seedling；
Fig. 7 is to take root after transplantation of seedlings 5 weeks.
Embodiment：
With reference to embodiment to further detailed description of the present invention, but the implementation of the present invention is not limited to this.Institute
The routine experimentation personnel for stating technical field take scope according to above present disclosure and each parameter, and this hair can be achieved
Bright purpose.The experimental method of unreceipted actual conditions in the following example, generally according to normal condition.
XMD culture mediums in following examples, its composition are as shown in table 1.
The constituent list of table 1XMD culture mediums
Described XMD culture mediums, every liter contains NH4NO3800mg、KNO3400mg、CaCl2·2H2O 200mg、Ca
(NO3)2·2H2O 200mg、MgSO4·7H2O 370mg、KH2PO4170mg、MnSO4·4H2O 22.3mg、ZnSO4·7H2O
8.6mg、H3BO39.3mg、KI 0.83mg、Na2MoO4·2H2O 0.25mg、CuSO4·5H2O 0.05mg、
CoCl20.025mg、FeSO4·7H2O 27.8mg、Na2·EDTA·H2O 37.3mg、Myo-Inositol 100mg、
Glycine 2mg, ThiamineHCl 0.1mg, Nicotinicacid 0.5mg, PyridoxineHCl 0.5mg,
Surplus is water, pH 5.8.Its compound method is the composition and content according to above-mentioned formula, and mentioned component is well mixed, and adjusts pH
5.8th, 121 DEG C of 15~20min of sterilizing, obtain XMD culture mediums, standby.
Embodiment 1：
A kind of high-efficiency in-vitro plant regeneration method of Prunus donarium adult fine individual plant ' little Qiao ' cherry, comprises the following steps：
(1) explant gathers：During the fine morning 10 in 3~May or so, from ' little Qiao ' cherry maternal plant (age of tree 10 years
More than adult select tree) on clip then give birth to robust growth, the semi-lignified young sprout of no disease and pests harm, moisturizing processing after take back experiment
Room, by the semi-lignified young sprout disleaf after collection, put 4 DEG C of refrigerators as explant after being cleaned up by the use of pure water and save backup.
Before gathering explant, branch is sprouted 4~5 times with carbendazim, Bravo alternating sprinkling every 10d.
(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 with sterilized water on platform, then gone out solution immersion 7min with 0.5% new gill of mass fraction, outwelled new gill and go out solution
Afterwards, with aseptic water washing 3 times, add in the alcohol water blend of volume fraction 70% and soak 20s, after outwelling alcohol water blend, then
The mercuric chloride solution of mass fraction 0.1% is added, 1~5min is soaked depending on the tender degree of children, constantly shakes therebetween, after outwelling mercuric chloride solution,
Aseptic water washing is used again 6 times.Aseptic filter paper blots sterilizable material surface water drops, with sterile razor blade cut off petiole, stem section both ends by
Behind the position of traumatic part, then stem section is cut into 2cm band axils and cuts off standby, to be sterilized band axil cut-out.
(3) axillary bud deriving：The band axil disinfected cut-out (explant) is inoculated on inducing culture and cultivated, cultivates bar
Part is：25 ± 2 DEG C, 60 μm of ol.m of intensity of illumination-2.s-1, light application time 12h/d, one explant of every bottle of inoculation, 3~4d it
Afterwards, petiole starts loose or dislocation, and axillary bud is sprouted, multiple axillary bud of axil energy eruption (Fig. 1, Fig. 2) of 15d or so, and inductivity is
100%, described inducing culture is that every liter containing 6-BA 1.5mg, NAA 0.1mg, sucrose 30g, agar 6g, (hormone is purchased
From Sigma-Aldrich companies), surplus is XMD culture mediums, and pH is 5.8 (its compound method is：Mentioned component is well mixed
Afterwards, pH value is adjusted, is sterilized standby), thus obtain axillary bud.
(4) Multiplying culture：The stem section that the axillary bud induced is cut into 1.0~1.5cm is transferred and cultivated on proliferated culture medium,
Condition of culture is：25 ± 2 DEG C, 60 μm of ol.m of intensity of illumination-2.s-1, light application time 12h/d, described proliferated culture medium, every liter
Containing 6-BA 1.0mg, NAA 0.05mg, sucrose 30g, agar 6g, surplus is XMD culture mediums, and pH is 5.8 (its compound method
For：By mentioned component it is well mixed after, adjust pH value, sterilizing is standby), after 15d, simple bud forms bud clump, and bud elongation growth is fast, propagation
Coefficient (Ya Gao≤2cm) 4.5 (Fig. 3, Fig. 4) are up to, thus obtain breeding seedling.
(5) culture of rootage：Cut from the healthy and strong effective bud of propagation seedling selection (bud height is more than 2cm) and be transferred to root media
Middle culture, condition of culture are：25 ± 2 DEG C, 60 μm of ol.m of intensity of illumination-2.s-1, light application time 12h/d, described culture of rootage
Base is, every liter containing NAA 1.0mg, sucrose 15g, agar 6g, surplus be XMD culture mediums, and (its compound method is pH for 5.8：Will
After mentioned component is well mixed, pH value is adjusted, sterilizing is standby), after 15d, the inductivity of adventitious root is 100%, and every plant averagely takes root
Count up to more than 5 (Fig. 5, Fig. 6).
(6) hardening and transplanting：The bottle seedling that will take root moves on in greenhouse adaptation 5~7d of external environment, then by bottle cap from
Standard-sized sheet hardening 1d is partly reached, carefully takes out the seedling in bottle after hardening, cleans and sticks the culture medium on seedling, be transplanted to and fill up mud
Charcoal soil:Yellow mud:Vermiculite=2:1:In the seedling-raising cup of 1 (volume ratio) mixed-matrix, each glass of one young plant of plantation, thickness of earth-fill cover is transplanted
Root system is just covered, is usually no more than 1cm, compresses matrix, is in close contact seedling root and matrix, final singling of being drenched after the completion of transplanting
Water.
(7) seedling culture：After transplanting, the management of moisture, fertilizer and anti-bacteria is carried out to seedling：
A, water management：Matrix keeps moistening to bottle seedling after the transfer, and relative air humidity is advisable 80%~90%, preceding 5d
25 DEG C or so of cultivation temperature can be controlled by epiphragma moisturizing, afterwards by moisturizing of spraying, cool canopy with 70% sunshade net shading；
B, 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；
C, stabilization to be grown, after growing sprouting and new root, intensity of illumination is stepped up, is routinely managed until carrying out full exposure
Reason, transplanting the latter moon survival rate is higher than 90% (Fig. 7), when seedling grows high to 15cm (' little Qiao ' cherry seedling), can be moved to big
Field cultivating large seedling.
Embodiment 2：
A kind of high-efficiency in-vitro plant regeneration method of Prunus donarium adult fine individual plant ' little Qiao ' cherry, comprises the following steps：
(1) explant gathers：During the fine morning 10 in 3~May or so, from ' little Qiao ' cherry maternal plant (age of tree 10 years
More than select tree) on clip then give birth to robust growth, the semi-lignified young sprout of no disease and pests harm, moisturizing processing after take back laboratory,
By the semi-lignified young sprout disleaf after collection, put 4 DEG C of refrigerators as explant after being cleaned up by the use of pure water and save backup.Collection
Before explant, branch is sprouted 4~5 times with carbendazim, Bravo alternating sprinkling every 10d.
(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 with sterilized water on platform, then gone out solution immersion 8min with 1% new gill of mass fraction, outwelled new gill and go out solution
Afterwards, with aseptic water washing 4 times, add in the alcohol water blend of volume fraction 75% and soak 30s, after outwelling alcohol water blend, then
The mercuric chloride solution of mass fraction 0.05% is added, 1~5min is soaked depending on the tender degree of children, constantly shakes therebetween, after outwelling mercuric chloride solution,
Aseptic water washing is used again 5 times.Aseptic filter paper blots sterilizable material surface water drops, with sterile razor blade cut off petiole, stem section both ends by
Behind the position of traumatic part, then stem section is cut into 2cm band axils and cuts off standby, to be sterilized band axil cut-out.
(3) axillary bud deriving：The band axil disinfected cut-out (explant) is inoculated on inducing culture and cultivated, cultivates bar
Part is：25 ± 2 DEG C, 60 μm of ol.m of intensity of illumination-2.s-1, light application time 12h/d, one explant of every bottle of inoculation, after 7d, leaf
Handle starts loose or dislocation, and axillary bud is sprouted, axil energy eruption one or more axillary bud of 15d or so, and inductivity 84% is described
Inducing culture be, every liter containing 6-BA 1.0mg, NAA 0.2mg, sucrose 25g, (hormone is purchased from Sigma- to agar 6g
Aldrich), surplus is XMD culture mediums, and pH is 6 (its compound method is：After mentioned component is well mixed, pH value is adjusted,
Sterilize standby), thus obtain axillary bud.
(4) Multiplying culture：The stem section that the axillary bud induced is cut into 1.0~1.5cm is transferred and cultivated on proliferated culture medium,
Condition of culture is：25 ± 2 DEG C, 60 μm of ol.m of intensity of illumination-2.s-1, light application time 12h/d, described proliferated culture medium, every liter
Containing 6-BA 0.5mg, NAA 0.1mg, sucrose 25g, agar 6g, surplus is XMD culture mediums, and pH is 6 (its compound method is：Will
After mentioned component is well mixed, pH value is adjusted, sterilizing is standby), after 15d, simple bud forms bud clump, and bud elongation growth is very fast, propagation system
Number (Ya Gao≤2cm) up to 3.2, thus obtain breeding seedling.
(5) culture of rootage：Cut from the healthy and strong effective bud of propagation seedling selection (bud height is more than 2cm) and be transferred to root media
Middle culture, condition of culture are：25 ± 2 DEG C, 60 μm of ol.m of intensity of illumination-2.s-1, light application time 12h/d, described culture of rootage
Base is, every liter containing NAA 0.5mg, sucrose 20g, agar 6g, surplus be XMD culture mediums, and (its compound method is pH for 6：Will be upper
State composition it is well mixed after, adjust pH value, sterilizing is standby), after 15d, the inductivity of adventitious root is 86%, and every plant of number of averagely taking root reaches
More than 5.
(6) hardening and transplanting：The bottle seedling that will take root moves on in greenhouse adaptation 5~7d of external environment, then by bottle cap from
Standard-sized sheet hardening 1d is partly reached, carefully takes out the seedling in bottle after hardening, cleans and sticks the culture medium on seedling, be transplanted to and fill up mud
Charcoal soil:Yellow mud:Vermiculite=2:1:In the seedling-raising cup of 1 (volume ratio) mixed-matrix, each glass of one young plant of plantation, thickness of earth-fill cover is transplanted
Root system is just covered, is usually no more than 1 centimetre, compresses matrix, is in close contact seedling root and matrix, final singling of being drenched after the completion of transplanting
Water.
(7) seedling culture：After transplanting, the management of moisture, fertilizer and anti-bacteria is carried out to seedling：
A, water management：Matrix keeps moistening to bottle seedling after the transfer, and relative air humidity is advisable 80%~90%, preceding 5d
25 DEG C or so of cultivation temperature can be controlled by epiphragma moisturizing, afterwards by moisturizing of spraying, cool canopy with 70% sunshade net shading；
B, 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；
C, stabilization to be grown, after growing sprouting and new root, intensity of illumination is stepped up, is routinely managed until carrying out full exposure
Reason, transplanting the latter moon survival rate is higher than 90%, when seedling grows high to 15cm (' little Qiao ' cherry seedling), can be moved to crop field cultivation
Seedlings.
Embodiment 3：
A kind of high-efficiency in-vitro plant regeneration method of Prunus donarium adult fine individual plant ' little Qiao ' cherry, comprises the following steps：
(1) explant gathers：During the fine morning 10 in 3~May or so, from ' little Qiao ' cherry maternal plant (age of tree 10 years
More than select tree) on clip then give birth to robust growth, the semi-lignified young sprout of no disease and pests harm, moisturizing processing after take back laboratory,
By the semi-lignified young sprout disleaf after collection, put 4 DEG C of refrigerators as explant after being cleaned up by the use of pure water and save backup.Collection
Before explant, branch is sprouted 4~5 times with carbendazim, Bravo alternating sprinkling every 10d.
(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 with sterilized water on platform, then gone out solution immersion 7min with 0.5% new gill of mass fraction, outwelled new gill and go out solution
Afterwards, with aseptic water washing 3 times, add in the alcohol water blend of volume fraction 70% and soak 30s, after outwelling alcohol water blend, then
The mercuric chloride solution of mass fraction 0.1% is added, 1~5min is soaked depending on the tender degree of children, constantly shakes therebetween, after outwelling mercuric chloride solution,
Aseptic water washing is used again 6 times.Aseptic filter paper blots sterilizable material surface water drops, with sterile razor blade cut off petiole, stem section both ends by
Behind the position of traumatic part, then stem section is cut into 2cm band axils and cuts off standby, to be sterilized band axil cut-out.
(3) axillary bud deriving：The band axil disinfected cut-out (explant) is inoculated on inducing culture and cultivated, cultivates bar
Part is：25 ± 2 DEG C, 60 μm of ol.m of intensity of illumination-2.s-1, light application time 12h/d, one explant of every bottle of inoculation, after 7d, leaf
Handle starts loose or dislocation, and axillary bud is sprouted, axil energy eruption one or more axillary bud of 15d or so, and inductivity 82% is described
Inducing culture be, every liter containing 6-BA 2.0mg, NAA 0.05mg, sucrose 40g, (hormone is purchased from Sigma- to agar 6g
Aldrich), surplus is XMD culture mediums, and pH is 5.8 (its compound method is：After mentioned component is well mixed, pH is adjusted
Value, sterilizing are standby), thus obtain axillary bud.
(4) Multiplying culture：The stem section that the axillary bud induced is cut into 1.0~1.5cm is transferred and cultivated on proliferated culture medium,
Condition of culture is：25 ± 2 DEG C, 60 μm of ol.m of intensity of illumination-2.s-1, light application time 12h/d, described proliferated culture medium, every liter
Containing 6-BA 1.5mg, NAA 0.05mg, sucrose 40g, agar 6g, surplus is XMD culture mediums, and pH is 5.8 (its compound method
For：By mentioned component it is well mixed after, adjust pH value, sterilizing is standby), after 15d, simple bud forms bud clump, and bud elongation growth is very fast, increases
Grow coefficient (Ya Gao≤2cm) up to 3.5, thus obtain breeding seedling.
(5) culture of rootage：Cut from the healthy and strong effective bud of propagation seedling selection (bud height is more than 2cm) and be transferred to root media
Middle culture, condition of culture are：25 ± 2 DEG C, 60 μm of ol.m of intensity of illumination-2.s-1, light application time 12h/d, described culture of rootage
Base is, every liter containing NAA 1.5mg, sucrose 15g, agar 6g, surplus be XMD culture mediums, and (its compound method is pH for 5.8：Will
After mentioned component is well mixed, pH value is adjusted, sterilizing is standby), after 15d, the inductivity of adventitious root is 79%, every plant of number of averagely taking root
Up to more than 4.
(6) hardening and transplanting：The bottle seedling that will take root moves on in greenhouse adaptation 5~7d of external environment, then by bottle cap from
Standard-sized sheet hardening 1d is partly reached, carefully takes out the seedling in bottle after hardening, cleans and sticks the culture medium on seedling, be transplanted to and fill up mud
Charcoal soil:Yellow mud:Vermiculite=2:1:In the seedling-raising cup of 1 (volume ratio) mixed-matrix, each glass of one young plant of plantation, thickness of earth-fill cover is transplanted
Root system is just covered, is usually no more than 1 centimetre, compresses matrix, is in close contact seedling root and matrix, final singling of being drenched after the completion of transplanting
Water.
(7) seedling culture：After transplanting, the management of moisture, fertilizer and anti-bacteria is carried out to seedling：
A, water management：Matrix keeps moistening to bottle seedling after the transfer, and relative air humidity is advisable 80%~90%, preceding 5d
25 DEG C or so of cultivation temperature can be controlled by epiphragma moisturizing, afterwards by moisturizing of spraying, cool canopy with 70% sunshade net shading；
B, 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；
C, stabilization to be grown, after growing sprouting and new root, intensity of illumination is stepped up, is routinely managed until carrying out full exposure
Reason, transplanting the latter moon survival rate is higher than 90%, when seedling grows high to 15cm (' little Qiao ' cherry seedling), can be moved to crop field cultivation
Seedlings.
Above-described embodiment is the preferable embodiment of the present invention, but embodiments of the present invention are not by above-described embodiment
Limitation, protection scope of the present invention are limited by appended claims, any change on the basis of the claims in the present invention
All it is protection scope of the present invention.
Claims (2)
- A kind of 1. high-efficiency in-vitro plant regeneration method of Prunus donarium adult fine individual plant ' little Qiao ' cherry, it is characterised in that including with Lower step：A, axillary bud deriving：In spring, semi-lignified young sprout is given birth on clip ' little Qiao ' cherry adult select tree maternal plant then as explant, Culture is inoculated on inducing culture after sterilized and obtains axillary bud, described inducing culture is, every liter containing 6-BA 1.0~ 0.05~0.2mg of 2.0mg, NAA, 25~40g of sucrose, the agar of constant, surplus are XMD culture mediums, and pH is 5.8~6.0；B, Multiplying culture：Axillary bud is cut and is inoculated on proliferated culture medium culture and obtains breeding seedling, described proliferated culture medium is, Every liter containing 0.5~1.5mg of 6-BA, 0.05~0.1mg of NAA, 25~40g of sucrose, constant agar, surplus be XMD culture Base, pH are 5.8~6.0；C, culture of rootage：Tender stem is cut from propagation seedling it is inoculated into root media culture and obtain rooted seedling, described takes root Culture medium is, every liter containing 0.5~1.5mg of NAA, 15~20g of sucrose, constant agar, surplus is XMD culture mediums, and pH is 5.8~6.0；Rooted seedling is moved on into hardening in greenhouse, then takes out seedling, cleans and sticks the culture medium on seedling, be transplanted to mud by volume Charcoal soil：Yellow mud：Vermiculite=2:1:In 1 mixed-matrix, training orientation is carried out, obtains ' little Qiao ' cherry seedling；Described XMD culture mediums, every liter contains NH4NO3 800mg、KNO3 400mg、CaCl2·2H2O 200mg、Ca(NO3)2· 2H2O 200mg、MgSO4·7H2O 370mg、KH2PO4 170mg、MnSO4·4H2O 22.3mg、ZnSO4·7H2O 8.6mg、 H3BO3 9.3mg、KI 0.83mg、Na2MoO4·2H2O 0.25mg、CuSO4·5H2O 0.05mg、CoCl2 0.025mg、 FeSO4·7H2O 27.8mg、Na2·EDTA·H2O 37.3mg, inositol 100mg, glycine 2mg, vitamin B1 0.1mg, cigarette Sour 0.5mg, vitamin B6 0.5mg, surplus are water, pH 5.8；It is in clip explant to give birth to semi-lignified young sprout as explant then on described clip ' little Qiao ' cherry adult select tree maternal plant Before body, every alternately ' little Qiao ' cherry adult select tree maternal plant tree body 4~5 times selected by sprinkling of 10d carbendazim, Bravo, then cut Take on ' little Qiao ' cherry maternal plant and to give birth to semi-lignified young sprout then as explant, disleaf, cleaned up by the use of pure water as explant, Low-temperature moisture preservation is standby；Be inoculated into after described sterilization on inducing culture is that the explant after sterilization is cut off into petiole, stem section two with sterile razor blade After holding injury, inoculate on inducing culture；Described sterilization is：Explant is soaked into 7~8min with the bromogeramine solution of mass fraction 0.5%~1%, outwelled new clean You go out after solution, soak 20~30s with aseptic water washing 3~4 times, then with the alcohol water blend of volume fraction 70~75%, fall After falling alcohol water blend, the addition mercuric chloride solution of mass fraction 0.05%~0.1%, the tender degree immersion 1 of foundation explant children~ 5min, during which constantly shake, outwell mercuric chloride solution, then with aseptic water washing 5~6 times, the explant after being sterilized.
- 2. the high-efficiency in-vitro plant regeneration method of Prunus donarium adult fine individual plant ' little Qiao ' cherry according to claim 1, its It is characterised by, described step b Multiplying culture is to cut axillary bud, is inoculated on proliferated culture medium and cultivates, axillary bud differentiation shape Into sprouting clump, the edible tender branch of bud height >=3cm in sprouting clump is cut into 1.0~1.5cm stem section, then transfer into new increasing Culture is grown in culture medium to obtain breeding seedling, described proliferated culture medium is, every liter containing 0.5~1.5mg of 6-BA, NAA0.05~ 0.1mg, 25~40g of sucrose, the agar of constant, surplus are XMD culture mediums, and pH is 5.8~6.0.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201510786966.7A CN105379621B (en) | 2015-11-16 | 2015-11-16 | A kind of high-efficiency in-vitro plant regeneration method of Prunus donarium adult fine individual plant " little Qiao " cherry |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201510786966.7A CN105379621B (en) | 2015-11-16 | 2015-11-16 | A kind of high-efficiency in-vitro plant regeneration method of Prunus donarium adult fine individual plant " little Qiao " cherry |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN105379621A CN105379621A (en) | 2016-03-09 |
| CN105379621B true CN105379621B (en) | 2017-12-29 |
Family
ID=55412883
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201510786966.7A Active CN105379621B (en) | 2015-11-16 | 2015-11-16 | A kind of high-efficiency in-vitro plant regeneration method of Prunus donarium adult fine individual plant " little Qiao " cherry |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN105379621B (en) |
Families Citing this family (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107135943A (en) * | 2017-04-20 | 2017-09-08 | 广州旺地园林工程有限公司 | A kind of winter cherry rapid propagation in vitro method |
| CN107006367B (en) * | 2017-04-20 | 2019-12-13 | 华南农业大学 | 'sunshine' cherry tissue culture rapid propagation method |
| 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 |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103416308A (en) * | 2013-08-08 | 2013-12-04 | 巴中市光雾山植物研究所 | Tissue culture rapid propagation method for wild sweet cherry trees |
| CN104521755A (en) * | 2014-12-18 | 2015-04-22 | 山东省果树研究所 | Culture medium for preventing etiolation of Yunan wild cherry test-tube plantlet and tissue culture method |
| CN105010144A (en) * | 2015-07-28 | 2015-11-04 | 福建世纪金花科技有限公司 | Double cherry tissue culture propagation method |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP3894914B2 (en) * | 2003-09-24 | 2007-03-22 | 独立行政法人科学技術振興機構 | Method for inducing adventitious buds of sweet cherry cultivars by high-temperature treatment |
-
2015
- 2015-11-16 CN CN201510786966.7A patent/CN105379621B/en active Active
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103416308A (en) * | 2013-08-08 | 2013-12-04 | 巴中市光雾山植物研究所 | Tissue culture rapid propagation method for wild sweet cherry trees |
| CN104521755A (en) * | 2014-12-18 | 2015-04-22 | 山东省果树研究所 | Culture medium for preventing etiolation of Yunan wild cherry test-tube plantlet and tissue culture method |
| CN105010144A (en) * | 2015-07-28 | 2015-11-04 | 福建世纪金花科技有限公司 | Double cherry tissue culture propagation method |
Non-Patent Citations (3)
| Title |
|---|
| In vitro propagation of Gisela 5(Prunus cerasus × P. canescens);Metka ŠIŠKO;《Agricultura》;20111231;第8卷;第31-34页 * |
| 垂枝早樱‘红枝垂’组培快繁试验;荣冬青等;《林业科技开发》;20081231;第22卷(第5期);第1-2节 * |
| 大岛樱的工厂化育苗技术;刘颂颂等;《广东林业科技》;19961231;第12卷(第4期);第2.1节 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN105379621A (en) | 2016-03-09 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN105475130B (en) | A kind of red cone isolated culture plant strain regeneration method | |
| CN106417015B (en) | A kind of Huaiji primulina tabacum tissue cultures and rapid propagation method | |
| CN104012417B (en) | High-efficiency and rapid micropropagation method for toxicodendron vernicifluum | |
| CN105613287B (en) | A kind of method bucket cotton rose tissue rapid propagation method for culturing seedlings | |
| CN103004604B (en) | Breeding method for vanda | |
| CN103430842A (en) | Quick propagation method of hybrid orchid tissue culture | |
| CN100429972C (en) | Tissue culture propagation process of late spring michelia | |
| CN105379621B (en) | A kind of high-efficiency in-vitro plant regeneration method of Prunus donarium adult fine individual plant " little Qiao " cherry | |
| CN106258960B (en) | A kind of orchid seed sprouting quick-breeding method | |
| CN107197746A (en) | A kind of mating system of China fir field excellent resources | |
| CN108739408A (en) | A kind of method for tissue culture improving the effective shoot differentiation quantity of great Ye spun gold Chinese catalpas | |
| CN102907325B (en) | Method for utilizing culture technology to produce Solomon turmeric and red tulip seedlings | |
| CN104604735B (en) | A kind of tissue culture and rapid propagation method of U.S.'s red autumnal leaves Lagerstroemia indica L. | |
| CN108142284A (en) | A kind of tissue culture and rapid propagation method of five leaflets maple | |
| CN106489737A (en) | A kind of culture medium of Hybrid Tea tissue cultures and method | |
| CN105340742B (en) | A kind of tissue culture and rapid propagation method of Yunnan cherry adult fine individual plant " Guangzhou " cherry | |
| CN103621399A (en) | Peony tissue culture method | |
| CN107135943A (en) | A kind of winter cherry rapid propagation in vitro method | |
| CN107667865A (en) | A kind of efficient subculture quick-breeding method of Ming River ceratostigma plumbaginoides Bunge | |
| CN107006367A (en) | A kind of ' sunlight ' cherry tissue culture and rapid propagation method | |
| CN108064689A (en) | A kind of method for tissue culture for seeing leaf begonia | |
| CN107155882A (en) | A kind of medicinal bletilla striata aseptic seeding fast seedling-cultivating method | |
| CN107182783A (en) | A kind of method of Hainan rhizomes of Panax notoginseng bastem portion regeneration induction plant | |
| CN103503771A (en) | Tissue culture and rapid propagation method for Australian hardenbergia violacea seedlings | |
| CN108812327B (en) | Culture solution and culture method for water culture propagation of chrysosporium |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| CB03 | Change of inventor or designer information | ||
| CB03 | Change of inventor or designer information |
Inventor after: Deng Xiaomei Inventor after: Ye Xiaoling Inventor after: Hu Xiaomin Inventor after: Xi Ruchun Inventor after: Zhu Jun Inventor after: Zheng Keyuan Inventor after: Zhang Xiaoming Inventor before: Deng Xiaomei Inventor before: Liu Xiangyuan Inventor before: Ye Xiaoling Inventor before: Xi Ruchun Inventor before: Hu Xiaomin Inventor before: Ye Chaohong Inventor before: Zhang Xiaoming |
|
| TA01 | Transfer of patent application right | ||
| TA01 | Transfer of patent application right |
Effective date of registration: 20170822 Address after: 510335, 483, five mountain road, Guangzhou, Guangdong, Tianhe District, China Applicant after: South China Agricultural University Applicant after: Guangzhou mangrove ecological science and Technology Co Ltd Applicant after: Guangzhou Wang garden Engineering Co., Ltd. Address before: 510335, 483, five mountain road, Guangzhou, Guangdong, Tianhe District, China Applicant before: South China Agricultural University Applicant before: GUANGDONG TRENDSEE CHERRY BIOSSOM AULACESE PARK CO., LTD. |
|
| GR01 | Patent grant | ||
| GR01 | Patent grant |