CN105475130B - A kind of red cone isolated culture plant strain regeneration method - Google Patents
A kind of red cone isolated culture plant strain regeneration method Download PDFInfo
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- CN105475130B CN105475130B CN201510833956.4A CN201510833956A CN105475130B CN 105475130 B CN105475130 B CN 105475130B CN 201510833956 A CN201510833956 A CN 201510833956A CN 105475130 B CN105475130 B CN 105475130B
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
- A01H4/00—Plant reproduction by tissue culture techniques ; Tissue culture techniques therefor
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Abstract
The invention belongs to the technical field of tissue culture of plant, disclose a kind of red cone isolated culture plant strain regeneration method.This method is, using red cone select tree seed as explant, by adventitious bud inducing, bud Multiplying culture and culture of rootage, to form intact plant by explant processing, minimal medium design, hormon type and concentration level ratio optimization.Develop the administrative skill for the seedling after transplantation of seedlings matrix and transplanting of taking root suitable for red cone tissue culture.It is blank that explant carries out tissue-culturing rapid propagation that the invention, which is compensated for using red cone select tree seed, provides a kind of stabilization, red cone isolated culture plant strain regeneration method, can carry out large-scale production, production cost is low, red cone seedling stalwartness, the neat and consistent cultivated.Red cone is Precious Timber Species, and afforestation area is big at present, and market high quality seedling is rare, and this technology implements that technical support and breeding guarantee can be provided for the economic benefit of red cone elite germplasm Rapid Popularization, the red cone artificial forest of raising, 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 red cone isolated culture plant strain regeneration side
Method.
Background technology
Red cone (Castanopsis hystrix A.DC) is subordinate to Fagaceae (Fagaceae) Castanopsis (Castanopsis),
Bearing tree height is the advantage composition tree of China's south subtropics and subtropical vegetation evergreen broadleaf forest up to 30m, diameter of a cross-section of a tree trunk 1.3 meters above the ground 1m or more
Kind, belong to South China important broad-leaved preciousness material of one's native land and multipurpose ecological public welfare forests seeds.Red cone natural distributed is in east longitude
95 ° 20 '~118 ° 00 ', 18 ° 30 '~25 ° 00 ' of north latitude concentrates on to main product Guangdong, Guangxi, south Fujian, circumferential distribution
South is saved in Hainan, south of Yunnan, Dong people and Hunan, Jiangxi etc..
Red cone has the good characteristics such as growth fast, material is excellent, adaptation is wide, high efficiency.Its trunk is logical straight, and material is hard, is in
Red, color and luster and beautiful texture, corrosion resistance is strong, does not crack, is indeformable, easy processing, is high-quality preciousness material, for building,
The use such as shipbuilding, top-grade furniture, floorboards of wood, military project articles for use, sports equipment.Seed is rich in starch, can be used for stir-frying and eating, feed and wine
Wine, kind is real, acorn-cup is rich in tannin, can obtain through refining tannin extract.Red cone woods rudiment power is strong, and coppice shoot growth is rapid, and primary afforestation can fell
10 times or more, Rational Management can make a profit upper a century.Red cone is thick with leaves, more resistance to concealment, and mixing natural disposition can be good, can be used as with material and water
Source conserves woods and carries out pure forest plantation, also can be that inferior woods is transformed, the Mixed forestation of ecological public welfare forests transformation.Multiple provinces of China at present
Such as red cone is elected in Guangdong, Guangxi, Fujian promotes to work as the fine tree species that regional economic value is high, development prospect is big,
Afforestation area is big, and popularizing application prospect is wide.
Red cone artificial forest cultivation nursery stock used mostly uses the mixed standing forest adopted seed seedling-raising, lead to after nursery stock and its afforestation at present
Individual differentiation is big, irregular, and then influences Afforestation Quality, yield and the economic benefit of artificial forest.Pass through scientific worker
The research of nearly more than ten years has selected the red cone fine individual plant of a batch, but due to selected excellent strain limited amount, the age of tree is young in addition,
Setting percentage is low, and harvested seed is far from meeting afforestation demand.Early-stage study shows red cone select tree no matter using grafting or skewer
Row breeding is injected, low reproduction rate exists simultaneously the different inclined hat phenomenon of degree, and institute's breeding nursery stock is difficult to use in production.With red cone
It is had been reported that though select tree sprouts branch progress group training research, the age of tree of select tree is not specified, may be reported due to genotype difference etc.
The tissue culture method in road has that Fiber differentiation is seriously polluted, and budding is slow for select tree tissue culture selected by this project, and aseptic strain is established tired
It is difficult；The problems such as bud of Multiplying culture is small and yellow, and growth is slow, gradually withered and yellow, proliferation times and low rooting rate, it is difficult to for producing.
The red cone precious reproducting tree species important as China, breeding nursery stock demand is huge, but vegetative propagation is difficult, and nursery stock supplies
It needs particularly thorny.This method using it is selected it is red cone select tree seed as explant, carry out group culturation rapid propagating technology research, be it is red cone this
Fine tree species realize breeding scale breeding, and then realize the important channel of industrialization development, have important research and production real
Border meaning.
In relation to using the tissue culture technique that red cone select tree seed is explant, there is not been reported.
Using the red cone select tree seed that the speed of growth is fast, dry type and material are excellent, resistance is strong as explant, pass through explant
Processing, minimal medium design, the optimization of hormone kind, concentration and its proportioning are established its isolated culture plant strain regeneration techniques, are reached
To explant Fiber differentiation pollution rate is low, the time is short, adventitious bud is more, the fast healthy and strong, appreciation rate of proliferation bud elongation growth and rooting rate
High purpose develops the administrative skill for the seedling after transplantation of seedlings matrix and transplanting of taking root suitable for red cone tissue culture.Pass through scale
Nursery is promoted, and technical support will be provided for the breeding of high quality seedling needed for the red cone artificial forest cultivation in China, to promoting red cone
Artificial forest economic benefit has extremely important meaning.
Invention content
In order to overcome the disadvantages and deficiencies of the prior art, fill up directly fast using red cone select tree seed as explant progress tissue culture
Numerous technological gap, the purpose of the present invention is to provide a kind of red cone isolated culture plant strain regeneration methods.It is indefinite that this method has
Bud induction is fast, adventitious bud is more, proliferation multiplying power is high, the sturdy elongation of bud is fast, rooting rate is high, rooted seedling well developed root system is healthy and strong, transplanting at
The features such as motility rate is high.
The purpose of the invention is achieved by the following technical solution：
A kind of red cone isolated culture plant strain regeneration method, including following operating procedure：
(1) DX minimal mediums, the DX bases are designed according to the nutritional need of red cone select tree seed physiology state, cultured in vitro
The formula of basal culture medium is as follows：720mg/L NH4NO3、480mg/L KNO3、600mg/L Ca(NO3)2·4H2O、370mg/L
MgSO4·7H2O、170mg/L KH2PO4、100mg/L KCl、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、0.05mg/L
NiSO4.6H2O、0.025mg/L CoCl2、27.8mg/L FeSO4·7H2O、37.3mg/L Na2·EDTA·H2O、120mg/L
Myo-Insitol (inositol), 2.0mg/L Glycine (glycine), 0.4mg/L ThiamineHCl (vitamin B1),
0.2mg/L Nicotinicacid (niacin), 0.2mg/L PyridoxineHCl (vitamin B6)；Its pH value is 5.8；
(2) explant prepares and handles；
(3) explant surface sterilization；
(4) bud induces：The seed disinfected is accessed in prepared inductive differentiation medium and is cultivated；The induction
Differential medium be in DX minimal mediums add 6-BA0.4~1.0mg/L, 0.2~0.5mg/L of KT, NAA0.02~
0.2mg/L, 25~40g/L of sucrose and carragheen 8g/L are prepared, and pH value is 5.8~6.0；
(5) Multiplying culture：By 25~30d of Fiber differentiation, bud of the length more than 2cm cut be transferred in proliferated culture medium into
Row Multiplying culture；20~30d of Multiplying culture is differentiated to form sprouting clump, the edible tender branch of Ya Gao≤3cm is cut into 1.0~
The stem section of 1.5cm is transferred into new proliferated culture medium, and proliferation times are 3~4.2 times；Expand by shoot proliferation culture repeatedly numerous
It grows；The proliferated culture medium is that 0.5~2.0mg/L of 6-BA, 0.2~0.5mg/L of KT, NAA are added in DX minimal mediums
0.05~0.2mg/L, 25~40g/L of sucrose and carragheen 8g/L are prepared, and pH value is 5.8~6.0；
(6) culture of rootage：By in proliferated culture medium bud height be more than that the tender stem of 1.5cm is cut, access root media in
It is cultivated, rooting rate is up to 80~90% after 20d；The root media is the addition NAA 0.5 in 1/2DX minimal mediums
~1.0mg/L, 15~20g/L of sucrose and agar 6g/L are prepared, and pH value is 5.8~6.0；
(7) acclimatization and transplants；
(8) seedling management is transplanted.
Step (1) the DX minimal mediums are sterilized 15~20 minutes under the conditions of 121 DEG C.
Step (2) explant prepares with processing to be that red cone select tree seed is peelled off acorn-cup in advance, in superclean bench
On with scalpel peel off interior exosper, pay attention to ensureing the complete of embryo.
Step (3) the explant surface sterilization is the wine for the seed concentration of volume percent 70~75% that will be handled well
Essence impregnates 20~30s, outwells after alcohol with aseptic water washing 1~2 time, be then added quality concentration of volume percent 0.05~
0.1% mercuric chloride solution impregnates 1~2min, constantly shakes therebetween, outwells mercuric chloride solution and uses aseptic water washing again 5~6 times.
It is 25 ± 2 DEG C that step (4) culture, which is in temperature, and intensity of illumination is 30 μm of ol.m-2.s-1, light application time is
It is cultivated in the culturing room of 8h/d；Axillary bud and adventitious bud are sprouted cultivating after 10d from cotyledonary node.
It is 25 ± 2 DEG C that step (5) culture, which is in temperature, and intensity of illumination is 60 μm of ol.m-2.s-1, light application time is
It is cultivated in the culturing room of 12h/d.
It is 25 ± 2 DEG C that step (6) culture, which is in temperature, and intensity of illumination is 80 μm of ol.m-2.s-1, light application time is
It is cultivated in the culturing room of 12h/d；The 1/2DX minimal mediums are by contained a great number of elements in DX minimal mediums, packet
Include NH4NO3、KNO3、Ca(NO3)2·4H2O、MgSO4·7H2O、KH2PO4Halve with the dosage of KCl, remaining components unchanged.
Step (7) described acclimatization and transplants are specifically according to the following steps：After culture of rootage 20d, the bottle seedling that will take root moves on to 60%
Shade greenhouse 7~10d of hardening, then by bottle cap from partly reaching standard-sized sheet hardening 1d, then seedling is taken out from culture bottle, is washed
The net culture medium sticked on root, is transplanted in the container for plant growth for filling up matrix, and transplanting depth just covers root system with earthing, compresses
Matrix makes nursery stock stablize, root water of drenching after the completion of transplanting；The matrix is by peat soil, perlite and coconut palm chaff by 3：1：1
Volume ratio mixes.
Step (8) the transplanting seedling management is according to the following steps：
(1) it uses epiphragma moisturizing within first week after transplanting, then opens film, matrix moistening, relative air humidity is kept to exist
95% or more, control 15~30 DEG C of cultivation temperature, cool canopy with 70% shading net shading；
(2) after transplanting on the day of using quality concentration of volume percent 0.1% Bravo or carbendazim solution spray it is small
Seedling, it is primary every spraying in 10 days later；15 days after transplanting, the composite fertilizer of quality concentration of volume percent 1~2 ‰ is sprayed, according to children
Seedling upgrowth situation sprayed primary every 10 days；
(3) it waits for that growth of seedling is stablized after transplanting, after growing sprouting and new root, is stepped up intensity of illumination, it is complete until carrying out
Illumination Routine Management, seedling culture to when 20cm high for afforesting.
Compared with prior art, the present invention has the following advantages and beneficial effects：Using the method for the present invention to red cone explant
The bud induced velocity of body is fast；Inductivity is up to 100%；Growth coefficient is big, up to 4.2；Clump bud is sturdy, proliferation bud elongation growth is fast,
Culture 30d buds are up to 4~5cm；Rooting rate is high, well developed root system, and up to 90%, mean elements is 3.4 pieces/plant；Transplanting survival rate
Height, up to 90% or more.By scale breeding, technical support will be provided for the red cone elite germplasm Rapid Popularization in China, to carry
It rises red cone artificial forest economic benefit and high quality seedling guarantee is provided, there is extremely important meaning.
Description of the drawings
Fig. 1 is that the bud of select tree seed asepsis material induces.
Fig. 2 is the bud clump that induced bud is formed.
Fig. 3 is the proliferation bud in proliferated culture medium.
Fig. 4 is the rooted seedling root system in root media.
Fig. 5 transplants for rooted seedling.
Specific implementation mode
With reference to embodiment, the present invention is described in further detail, and embodiments of the present invention are not limited thereto.
DX minimal mediums are designed according to the nutritional need of red cone select tree seed physiology state, cultured in vitro, are implemented below
The formula of the DX minimal mediums arrived involved in example is as follows：720mg/L NH4NO3、480mg/L KNO3、600mg/L Ca
(NO3)2·4H2O、370mg/L MgSO4·7H2O、170mg/L KH2PO4、100mg/L KCl、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、0.05mg/L NiSO4.6H2O、0.025mg/L CoCl2、27.8mg/L FeSO4·7H2O、
37.3mg/L Na2·EDTA·H2O, 120mg/L Myo-Insitol (inositol), 2.0mg/L Glycine (glycine),
0.4mg/L ThiamineHCl (vitamin B1), 0.2mg/L Nicotinicacid (niacin), 0.2mg/L
PyridoxineHCl (vitamin B6)；Its pH value is 5.8.
The 1/2DX minimal mediums are by contained a great number of elements (NH4NO3, KNO3, Ca in DX minimal mediums
(NO3) 24H2O, MgSO47H2O, KH2PO4, KCl) dosage halve, remaining components unchanged.
Embodiment one realizes the isolated culture plant strain regeneration of red cone using following steps：
(1) adventitious bud inducing of red cone select tree seed
Explant prepares and processing：Selected select tree seed is peelled off into acorn-cup with secateurs in advance, with solution on superclean bench
Cut open cutter peels off interior exosper, pays attention to ensureing the complete of embryo.
Explant surface sterilization：First seed is scrubbed with sterile water on superclean bench, then uses percent by volume
Then quality concentration of volume percent is added in the alcohol of concentration 75%, soaking time 20s, later aseptic water washing 1 time
0.05% mercuric chloride solution impregnates 2min, constantly shakes therebetween, outwells mercuric chloride solution aseptic water washing 6 times.Aseptic filter paper blots
Sterilizable material surface water drops are spare.
Bud induces：The embryo disinfected is inoculated into inducing culture DX+0.8mg/L 6-BA+0.2mg/L KT+
0.02mg/L NAA+ sucrose 30g/L+ carragheens 8g/L (hormone is purchased from Sigma-Aldrich companies), one explant of every bottle of inoculation
Body.After 2~3d, embryo starts to sprout, and after a week, epicotyl is up to 0.2cm, the multiple budlets of eruption (Fig. 1, Fig. 2) after 20 days,
Inductivity is 100%.
(2) clump bud is proliferated
The stem section that the axillary bud induced is cut into 1.0~1.5cm is transferred into proliferated culture medium.Proliferation culture medium formula is DX
It is cultivated in the proliferated culture medium of+6-BA 0.5mg/L+KT 0.2mg/L+NAA 0.05mg/L+ sucrose 30g/L+ carragheens 8g/L,
After 4 weeks, simple bud forms bud clump, and bud elongation growth is fast, and growth coefficient is up to 4.2, effective bud (Ya Gao≤2cm) up to 3.32/clump (figure
3)。
(3) culture of rootage
Healthy and strong effective bud is chosen, the rooting induction of 1/2DX+NAA 0.5mg/L+ sucrose 15g/L+ agar 6g/L is transferred to
It is cultivated in culture medium, after 15d, the inductivity of adventitious root is 85%, 3.4/plant of mean elements (Fig. 4).
(4) tissue-cultured seedling transplanting and management
By 3：1：Peat soil, perlite and coconut palm chaff are uniformly mixed by 1 volume ratio, container bag are packed into, with quality volume hundred
Divide 3 ‰ disinfecting solution of potassium permanganate of specific concentration for use；It is that temperature is controlled at 25 DEG C to transplant environmental condition, and 70% obscurity is empty
Greenhouse of the gas relative humidity 95% or more；The bottle seedling of taking root of culture of rootage 20d is moved on to and adapts to the external world in 60% shade greenhouse
Then environment 7d carefully takes out tissue-cultured seedling by bottle cap from standard-sized sheet hardening 1d is partly reached, clean the culture sticked on root
Base is transplanted in container bag, and a young plant is planted per plastic bag cultivation, and transplanting thickness of earth-fill cover just covers root system and stablizes nursery stock, compresses matrix, move
Root water is irrigated after the completion of planting；The last week uses epiphragma moisturizing after transplanting, then opens film, keeps matrix moistening；It is transplanting
The same day sprays seedling using Bravo (or carbendazim) solution of quality concentration of volume percent 0.1% afterwards, later every 10 days
It sprays once, prevents germ from growing；15 days after transplanting, start to spray 2 ‰ composite fertilizer of quality concentration of volume percent, according to seedling
Upgrowth situation sprayed primary every 10 days；It waits for that growth of seedling is stablized, after growing sprouting and new root, is stepped up intensity of illumination, directly
To full exposure Routine Management is carried out, when seedling culture is to 20cm high, can be used for afforesting.Transplanting survival rate is higher than 90% (Fig. 5).
Embodiment two realizes the isolated culture plant strain regeneration of red cone using following steps：
(1) adventitious bud inducing of red cone select tree seed
Explant prepares and processing：Selected select tree seed is peelled off into acorn-cup with secateurs in advance, with solution on superclean bench
Cut open cutter peels off interior exosper, pays attention to ensureing the complete of embryo.
Explant surface sterilization：First seed is scrubbed with sterile water on superclean bench, then uses percent by volume
Then quality concentration of volume percent is added in the alcohol of concentration 72%, soaking time 30s, later aseptic water washing 2 times
0.1% mercuric chloride solution impregnates 1min, constantly shakes therebetween, outwells mercuric chloride solution aseptic water washing 5 times.Aseptic filter paper blots
Sterilizable material surface water drops are spare.
Bud induces：The embryo disinfected is inoculated into inducing culture DX+0.4mg/L 6-BA+0.2mg/L KT+
0.1mg/L NAA+ sucrose 25g/L+ carragheens 8g/L (hormone is purchased from Sigma-Aldrich companies), one explant of every bottle of inoculation
Body.After 8~10d, embryo starts to sprout, and after two weeks, epicotyl reaches 0.2cm, and the multiple budlets of eruption, inductivity are after 30 days
85%.
(2) clump bud is proliferated
The stem section that the axillary bud induced is cut into 1.0~1.5cm is transferred into proliferated culture medium.Proliferation culture medium formula is DX
It is cultivated in the proliferated culture medium of+6-BA 1.0mg/L+KT 0.2mg/L+NAA 0.1mg/L+ sucrose 25g/L+ carragheens 8g/L, 4
Zhou Hou, simple bud form bud clump, and bud elongation growth is fast, and growth coefficient is up to 3, effective bud (bud height≤2cm) up to 2.82/clump.
(3) culture of rootage
Healthy and strong effective bud is chosen, the rooting induction of 1/2DX+NAA 0.7mg/L+ sucrose 20g/L+ agar 6g/L is transferred to
It is cultivated in culture medium, after 15d, the inductivity of adventitious root is 80%, 2.5/plant of mean elements.
(4) tissue-cultured seedling transplanting and management
By 3：1：Peat soil, perlite and coconut palm chaff are uniformly mixed by 1 volume ratio, container bag are packed into, with quality volume hundred
Divide 3 ‰ disinfecting solution of potassium permanganate of specific concentration for use；It is that temperature is controlled at 15 DEG C to transplant environmental condition, and 70% obscurity is empty
Greenhouse of the gas relative humidity 95% or more；The bottle seedling of taking root of culture of rootage 20d is moved on to and adapts to the external world in 60% shade greenhouse
Then environment 8d carefully takes out tissue-cultured seedling by bottle cap from standard-sized sheet hardening 1d is partly reached, clean the culture sticked on root
Base is transplanted in container bag, and a young plant is planted per plastic bag cultivation, and transplanting thickness of earth-fill cover just covers root system and stablizes nursery stock, compresses matrix, move
Root water is irrigated after the completion of planting；The last week uses epiphragma moisturizing after transplanting, then opens film, keeps matrix moistening；It is transplanting
The same day sprays seedling using Bravo (or carbendazim) solution of quality concentration of volume percent 0.1% afterwards, later every 10 days
It sprays once, prevents germ from growing；15 days after transplanting, start to spray 1 ‰ composite fertilizer of quality concentration of volume percent, according to seedling
Upgrowth situation sprayed primary every 10 days；It waits for that growth of seedling is stablized, after growing sprouting and new root, is stepped up intensity of illumination, directly
To full exposure Routine Management is carried out, when seedling culture is to 20cm high, can be used for afforesting.Transplanting survival rate is higher than 82% (Fig. 5).
Embodiment three realizes the isolated culture plant strain regeneration of red cone using following steps：
(1) adventitious bud inducing of red cone select tree seed
Explant prepares and processing：Selected select tree seed is peelled off into acorn-cup with secateurs in advance, with solution on superclean bench
Cut open cutter peels off interior exosper, pays attention to ensureing the complete of embryo.
Explant surface sterilization：First seed is scrubbed with sterile water on superclean bench, then uses percent by volume
Then quality concentration of volume percent is added in the alcohol of concentration 70%, soaking time 30s, later aseptic water washing 1 time
0.1% mercuric chloride solution impregnates 2min, constantly shakes therebetween, outwells mercuric chloride solution aseptic water washing 6 times.Aseptic filter paper blots
Sterilizable material surface water drops are spare.
Bud induces：The embryo disinfected is inoculated into inducing culture DX+1.0mg/L 6-BA+0.5mg/L KT+
0.2mg/L NAA+ sucrose 40g/L+ carragheens 8g/L (hormone is purchased from Sigma-Aldrich companies), one explant of every bottle of inoculation
Body.After 6~7d, embryo starts to sprout, and after 10 days, epicotyl reaches 0.2cm, the multiple budlets of eruption after 30 days, inductivity 90%.
(2) clump bud is proliferated
The stem section that the axillary bud induced is cut into 1.0~1.5cm is transferred into proliferated culture medium.Proliferation culture medium formula is DX
It is cultivated in the proliferated culture medium of+6-BA 2.0mg/L+KT 0.5mg/L+NAA 0.2mg/L+ sucrose 40g/L+ carragheens 8g/L, 4
Zhou Hou, simple bud form bud clump, and bud elongation growth is fast, and growth coefficient is up to 3.5, effective bud (bud height≤2cm) up to 2.52/clump.
(3) culture of rootage
Healthy and strong effective bud is chosen, the rooting induction of 1/2DX+NAA 1.0mg/L+ sucrose 15g/L+ agar 6g/L is transferred to
It is cultivated in culture medium, after 15d, the inductivity of adventitious root is 82%, 3/plant of mean elements.
(4) tissue-cultured seedling transplanting and management
By 3：1：Peat soil, perlite and coconut palm chaff are uniformly mixed by 1 volume ratio, container bag are packed into, with quality volume hundred
Divide 3 ‰ disinfecting solution of potassium permanganate of specific concentration for use；It is that temperature is controlled at 30 DEG C to transplant environmental condition, and 70% obscurity is empty
Greenhouse of the gas relative humidity 95% or more；The bottle seedling of taking root of culture of rootage 20d is moved on to and adapts to the external world in 60% shade greenhouse
Then environment 10d carefully takes out tissue-cultured seedling by bottle cap from standard-sized sheet hardening 1d is partly reached, clean the culture sticked on root
Base is transplanted in container bag, and a young plant is planted per plastic bag cultivation, and transplanting thickness of earth-fill cover just covers root system and stablizes nursery stock, compresses matrix, move
Root water is irrigated after the completion of planting；The last week uses epiphragma moisturizing after transplanting, then opens film, keeps matrix moistening；It is transplanting
The same day sprays seedling using Bravo (or carbendazim) solution of quality concentration of volume percent 0.1% afterwards, later every 10 days
It sprays once, prevents germ from growing；15 days after transplanting, start to spray 2 ‰ composite fertilizer of quality concentration of volume percent, according to seedling
Upgrowth situation sprayed primary every 10 days；It waits for that growth of seedling is stablized, after growing sprouting and new root, is stepped up intensity of illumination, directly
To full exposure Routine Management is carried out, when seedling culture is to 20cm high, can be used for afforesting.Transplanting survival rate is higher than 85% (Fig. 5).
The above embodiment is a preferred embodiment of the present invention, but embodiments of the present invention are not by above-described embodiment
Limitation, it is other it is any without departing from the spirit and principles of the present invention made by changes, modifications, substitutions, combinations, simplifications,
Equivalent substitute mode is should be, is included within the scope of the present invention.
Claims (7)
1. a kind of red cone isolated culture plant strain regeneration method, it is characterised in that including following operating procedure：
(1) DX minimal mediums are designed according to the nutritional need of red cone select tree seed physiology state, cultured in vitro, which trains substantially
The formula for supporting base is as follows：720mg/L NH4NO3、480mg/L KNO3、600mg/L Ca(NO3)2·4H2O、370mg/L
MgSO4·7H2O、170mg/L KH2PO4、100mg/L KCl、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、0.05mg/L
NiSO4· 6H2O、0.025mg/L CoCl2、27.8mg/L FeSO4·7H2O、37.3mg/L Na2·EDTA·H2O、
120mg/L Myo-Insitol、2.0mg/L Glycine、0.4mg/L Thiamine·HCl、0.2mg/L Nicotinic·
acid、0.2mg/L Pyridoxine·HCl；Its pH value is 5.8；
(2) explant prepares and handles：Red cone select tree seed is peelled off into acorn-cup in advance, is peelled off with scalpel on superclean bench
Interior exosper pays attention to ensureing the complete of embryo；
(3) explant surface sterilization：The alcohol of the seed handled well concentration of volume percent 70~75% is impregnated 20~
30s, aseptic water washing is used 1~2 time after outwelling alcohol, and it is molten that quality 0.05~0.1% mercuric chloride of concentration of volume percent is then added
Liquid impregnates 1~2min, constantly shakes therebetween, outwells mercuric chloride solution and uses aseptic water washing again 5~6 times；
(4) bud induces：The seed disinfected is accessed in prepared inductive differentiation medium and is cultivated；The induction differentiation
Culture medium be added in DX minimal mediums 6-BA 0.4~1.0mg/L, 0.2~0.5mg/L of KT, NAA 0.02~
0.2mg/L, 25~40g/L of sucrose and carragheen 8g/L are prepared, and pH value is 5.8~6.0；
(5) Multiplying culture：By 25~30d of Fiber differentiation, bud of the length more than 2cm, which cuts to be transferred in proliferated culture medium, to be increased
Grow culture；20~30d of Multiplying culture is differentiated to form sprouting clump, the edible tender branch of bud height >=3cm is cut into 1.0~1.5cm
Stem section transfer into new proliferated culture medium, proliferation times are 3~4.2 times；By shoot proliferation culture expanding propagation repeatedly；Institute
It is that 0.5~2.0mg/L of 6-BA, 0.2~0.5mg/L of KT, NAA 0.05 are added in DX minimal mediums to state proliferated culture medium
~0.2mg/L, 25~40g/L of sucrose and carragheen 8g/L are prepared, and pH value is 5.8~6.0；
(6) culture of rootage：By in proliferated culture medium bud height be more than that the tender stem of 1.5cm is cut, access root media in carry out
Culture, rooting rate is up to 80~90% after 20d；The root media be in 1/2DX minimal mediums add NAA0.5~
1.0mg/L, 15~20g/L of sucrose and agar 6g/L are prepared, and pH value is 5.8~6.0；The 1/2DX minimal mediums
For by contained a great number of elements in DX minimal mediums, including NH4NO3、KNO3、Ca(NO3)2·4H2O、MgSO4·7H2O、KH2PO4
Halve with the dosage of KCl, remaining components unchanged；
(7) acclimatization and transplants；
(8) seedling management is transplanted.
2. a kind of red cone isolated culture plant strain regeneration method according to claim 1, it is characterised in that：Step (1) is described
DX minimal mediums are sterilized 15~20 minutes under the conditions of 121 DEG C.
3. a kind of red cone isolated culture plant strain regeneration method according to claim 1, it is characterised in that：Step (4) is described
It is 25 ± 2 DEG C that culture, which is in temperature, and intensity of illumination is 30 μm of ol.m-2.s-1, light application time be 8h/d culturing room in trained
It supports；Axillary bud and adventitious bud are sprouted cultivating after 10d from cotyledonary node.
4. a kind of red cone isolated culture plant strain regeneration method according to claim 1, it is characterised in that：Step (5) is described
It is 25 ± 2 DEG C that culture, which is in temperature, and intensity of illumination is 60 μm of ol.m-2.s-1, light application time be 12h/d culturing room in trained
It supports.
5. a kind of red cone isolated culture plant strain regeneration method according to claim 1, it is characterised in that：Step (6) is described
It is 25 ± 2 DEG C that culture, which is in temperature, and intensity of illumination is 80 μm of ol.m-2.s-1, light application time be 12h/d culturing room in trained
It supports.
6. a kind of red cone isolated culture plant strain regeneration method according to claim 1, it is characterised in that：Step (7) is described
Acclimatization and transplants are specifically according to the following steps：After culture of rootage 20d, the bottle seedling that will take root moves on to 60% shade greenhouse 7~10d of hardening,
Then by bottle cap from partly reaching standard-sized sheet hardening 1d, then seedling taken out from culture bottle, cleans the culture medium sticked on root,
It is transplanted in the container for plant growth for filling up matrix, transplanting depth just covers root system with earthing, compresses matrix, nursery stock is made to stablize, and moves
Plant root water of drenching after the completion；The matrix is by peat soil, perlite and coconut palm chaff by 3：1：1 volume ratio mixes.
7. a kind of red cone isolated culture plant strain regeneration method according to claim 1, it is characterised in that：Step (8) is described
Transplant seedling management according to the following steps：
(1) it uses epiphragma moisturizing within first week after transplanting, then opens film, keep matrix moistening, relative air humidity is 95%
More than, control 15~30 DEG C of cultivation temperature, cool canopy with 70% shading net shading；
(2) after transplanting on the day of using quality concentration of volume percent 0.1% Bravo or carbendazim solution spray seedling, it
It is primary every spraying in 10 days afterwards；15 days after transplanting, the composite fertilizer of quality concentration of volume percent 1~2 ‰ is sprayed, according to growth of seedling
Situation sprayed primary every 10 days；
(3) it waits for that growth of seedling is stablized after transplanting, after growing sprouting and new root, is stepped up intensity of illumination, until carrying out full exposure
Routine Management, seedling culture to when 20cm high for afforesting.
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| CN106069769A (en) * | 2016-06-27 | 2016-11-09 | 广东省林业科学研究院 | A kind of sterilization method of the outer implant of red cone |
| CN106472315A (en) * | 2016-10-17 | 2017-03-08 | 柳州玲通科技有限责任公司 | A kind of Semen Arecae Ke's initial culture bud abductive approach |
| CN106359105A (en) * | 2016-10-17 | 2017-02-01 | 柳州玲通科技有限责任公司 | Inducing method for primary culture buds of lithocarpus hancei |
| CN106258990A (en) * | 2016-10-17 | 2017-01-04 | 柳州玲通科技有限责任公司 | A kind of Mei Yeke initial culture bud inducement method |
| CN106417031A (en) * | 2016-10-17 | 2017-02-22 | 柳州玲通科技有限责任公司 | Rootage induction method of lithocarpus amygdalifolius tissue culture seedlings |
| CN106258991A (en) * | 2016-10-17 | 2017-01-04 | 柳州玲通科技有限责任公司 | A kind of duricrust Ke's tissue cultured seedling root induction method |
| CN106359106A (en) * | 2016-10-17 | 2017-02-01 | 柳州玲通科技有限责任公司 | Lithocarpus oleaefolius tissue culture seedling rooting inducing method |
| CN106472316A (en) * | 2016-10-17 | 2017-03-08 | 柳州玲通科技有限责任公司 | A kind of Quercus acutissima initial culture bud abductive approach |
| CN106472314A (en) * | 2016-10-17 | 2017-03-08 | 柳州玲通科技有限责任公司 | A kind of dead ears Ke's tissue cultured seedling root induction method |
| CN106472313A (en) * | 2016-10-17 | 2017-03-08 | 柳州玲通科技有限责任公司 | A kind of Quercus acutissima tissue cultured seedling root induction method |
| CN106258989A (en) * | 2016-10-17 | 2017-01-04 | 柳州玲通科技有限责任公司 | A kind of Semen Arecae Ke's tissue cultured seedling root induction method |
| CN106879463A (en) * | 2017-02-14 | 2017-06-23 | 唐春艳 | Red cone aseptic explant is prepared and initial bud abductive approach |
| CN106797889A (en) * | 2017-02-15 | 2017-06-06 | 唐春艳 | A kind of red cone spray tissue culture propagation method |
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