CN105123519B - Ground is by the method for silvery birch tissue cultures - Google Patents
Ground is by the method for silvery birch tissue cultures Download PDFInfo
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Abstract
The invention discloses a kind of by the method for silvery birch tissue cultures.The method is that the explant after sterilization is seeded in clump bud inducement cultivation base to induce clump bud；Clump bud is inoculated into subculture multiplication medium again to continue to cultivate；Then it is seeded in root media and is taken root, hardening and field planting management is carried out after taking root.The inventive method acquisition ground is high by the yield of silvery birch clone nursery stock, and the time is fast, and the nursery stock for being obtained is healthy and strong, and these nursery stocks are lacked with solving the problems, such as planting industry by silvery birch.
Description
Technical field
The present invention relates to the method for Plant Tissue Breeding, and in particular to a kind ofly by the method for silvery birch tissue cultures.
Background technology
Silvery birch (Grevillea) is that Proteaceae (Proteaceae) can burst forth the plant of large-scale inflorescence, originates in Australia big
Sulawesi Island in the middle part of Leah, New Guinea, New Caledonia and Indonesia.Silvery birch is that Australian native country is planted
The nearly the third-largest seeds for being inferior to yearning between lovers and eucalyptus, have a very wide distribution, from the tropical rain forest of annual rainfall 3000mm to annual rainfall in thing
The desert area of 200mm, is all distributed from high mountain to coastal and arid inland.Genotype is varied, about 360 kinds, product
Kind of change is from the ground less than 0.5m by silvery birch to 1~5m shrubs and the up to arbor of 35m.Silvery birch easily realize interspecific hybridization and from
So variation, therefore produce naturally or artificially cultivated hundreds of variety and cenospecies.
Ground by silvery birch is famous drought-resistant seeds, and in being distributed in Australia mostly, western desert area, this area is normal
Year drought, therefore ground has very strong drought-resistant ability by many kinds of silvery birch.There is many on ground in silvery birch root structure
Tiny intensive fibrous root, this be also long term survival it is barren on the spot under evolution result, such structure is conducive to absorbing barren
Nutrient in soil, therefore ground is also barren-resistant seeds by silvery birch.Plantation ground only needs to apply the slow-release fertilizer of low fertilizer efficiency by silvery birch, or
Person does not apply fertilizer.Therefore, ground has the characteristics of managing and protecting low cost by silvery birch.
Ground is raceme by silvery birch, and its flower pattern is peculiar, is in mostly hairbrush shape or spider shape, and flower-shape is big, and pattern is more, from
Blood orange yellow has to yellow fraction, milky white or even green, and the florescence is long, and some kind whole years bloom, and ground is leaf abundant by silvery birch, from pin
Shape, blade is shallow to be split and drastic crack, and leaf color is mostly dark green, grayish green, has ornamental value very high on gardening.Ground is adapted to strong by silvery birch
Degree is pruned, and plastic to produce the gardening for coming in every shape tree-like.Ground by silvery birch be grafted onto on the stock of shrub silvery birch and arbor silvery birch into
Motility rate is high, can cultivate new weeping branch silvery birch, therefore ground is widely used in courtyard greening and scape by Australia by silvery birch
In sight engineering.Ground, containing abundant nectar, can provide food by silvery birch for the mankind and birds, honeybee class, therefore ground is have by silvery birch
The trick bird of name and the seeds kept pet.
A large amount of grown places are also immature by the technology of silvery birch nursery stock at present for China, it is difficult to accomplish intensive, the production managed
High efficiency, cannot also meet market over the ground by the wilderness demand of silvery birch seedling.Method for building up is simple, rooting rate is high, survival rate
Ground high will be played an important role by the popularization and application of silvery birch over the ground by silvery birch method for tissue culture.
The content of the invention
In order to solve above-mentioned problem, the present invention is introducing projects " introduced by silvery birch new varieties and raising technology "
Support under carried out ground by silvery birch Study on tissue culture, establish the ground of maturation by silvery birch tissue culture technique platform and factory
Change the technological process of nursery, and 30,000 plants of ground of successful reproduction by silvery birch tissue-cultured seedling.
It is an object of the invention to provide a kind of by the method for silvery birch tissue cultures.
The technical solution used in the present invention is：
A kind ofly by the method for silvery birch tissue cultures, comprise the following steps：
1) collection of explant；
2) sterilization of explant；
3) clump bud Fiber differentiation：Explant after upper step is sterilized is seeded in clump bud inducement cultivation base, and Fiber differentiation 3~
5 months, period need to be forwarded to new clump bud inducement cultivation base 3~5 times, and explant can be induced multiple clump buds；
4) shoot proliferation culture：Upper one-step inducing is gone out into the ground of clump bud by silvery birch tissue culture plant inoculation to subculture multiplication medium
In, often cultivate to be forwarded in new subculture multiplication medium ground by silvery birch propagation seedling in 28~32 days and continue to cultivate；
5) rooting induction culture：By in Multiplying culture highly up to more than 2cm simple bud cut access root media in, training
Support 10~15 days；
6) hardening：Enter the hardening stage after at least 60% propagation seedling starts to take root, the ground of acquisition is taken root by silvery birch
Tissue-cultured seedling is in 3000~6000Lx of light intensity, 26~30 DEG C of temperature condition lower refining seedling 15~20 days；
7) transplant and manage：By in the Nursery stock transplanting after hardening to transplanting medium, field planting management is carried out.
Further, step 1) in time of explant collection be June to September, gather 25~30cm portions of new coppice shoot top tip
Point, and cut off blade.
Further, step 2) in the specific method disinfected of explant be：The branch of collection is cleaned up, is sheared
Into 3~4cm it is long, with 1~2 stem section of axil, with the HgCl of 0.08~0.12%v/v in gnotobasis2Soaking disinfection 3~
7min, period, constantly shake ensured that thimerosal is fully contacted with branch, then clean with sterile water wash, finally by stem section two ends
0.4~0.6cm is respectively cut, middle stem section containing axil is left as aseptic explant.
Further, step 3) in the formula of clump bud inducement cultivation base be：412~413mg/L NH4NO3, 1898~
1902mg/L KNO3, 84.5~85.5mg/L KH2PO4, 369~371mg/L MgSO4·7HO2, 131~133mg/L
CaCl2·2HO2, 22~22.5mg/L MnSO4·4HO2, 8.4~8.8mg/L ZnSO4·7HO2, 6~6.4mg/L H3BO3、
0.22~0.27mg/L Na2MoO4·2HO2, 27.5~28mg/L FeSO4·7HO2, 37~37.5mg/L Na2·EDTA·
2HO2, 0.8~0.85mg/L KI, 0.023~0.027mg/L CuSO4·5HO2, 0.023~0.027mg/L CoCl2·
6HO2, 1.8~2.2mg/L glycine, 0.08~0.12mg/L thiamine hydrochlorides, 0.48~0.52mg/L puridoxine hydrochlorides,
0.48~0.52mg/L nicotinic acid, 99~101mg/L inositols, 0.8~1.2g/L PVP, 0.48~0.52mg/L 6-BA, 0.048
~0.052mg/L IBA, 27~32g/L sucrose, 7~8g/L carragheens, pH5.75~5.85.
Further, step 4) in the formula of subculture multiplication medium be：412~413mg/L NH4NO3, 1898~
1902mg/L KNO3, 84.5~85.5mg/L KH2PO4, 369~371mg/L MgSO4·7HO2, 131~133mg/L
CaCl2·2HO2, 22~22.5mg/L MnSO4·4HO2, 8.4~8.8mg/L ZnSO4·7HO2, 6~6.4mg/L H3BO3、
0.22~0.27mg/L Na2MoO4·2HO2, 27.5~28mg/L FeSO4·7HO2, 37~37.5mg/L Na2·EDTA·
2HO2, 0.8~0.85mg/L KI, 0.023~0.027mg/L CuSO4·5HO2, 0.023~0.027mg/L CoCl2·
6HO2, 1.8~2.2mg/L glycine, 0.08~0.12mg/L thiamine hydrochlorides, 0.48~0.52mg/L puridoxine hydrochlorides,
0.48~0.52mg/L nicotinic acid, 99~101mg/L inositols, 0.8~1.2g/L PVP, 0.2~0.3mg/L 6-BA, 0.048~
0.052mg/L IBA, 27~32g/L sucrose, 7~8g/L carragheens, pH5.75~5.85.
Further, step 5) in the formula of rooting induction culture medium be：412~413mg/LNH4NO3, 473~477mg/
L KNO3, 42~43mg/L KH2PO4, 92~93mg/L MgSO4·7HO2, 108~112mg/L CaCl2·2HO2, 11~
11.5mg/L MnSO4·4HO2, 4~4.5mg/L ZnSO4·7HO2, 2.8~3.4mg/L H3BO3, 0.12~0.13mg/L
Na2MoO4·2HO2, 13.5~14.5mg/L FeSO4·7HO2, 18.3~18.9mg/L Na2·EDTA·2HO2, 0.41~
0.42mg/L KI, 0.012~0.013mg/L CuSO4·5HO2, 0.012~0.013mg/L CoCl2·6HO2, 1.8~
2.2mg/L glycine, 0.08~0.12mg/L thiamine hydrochlorides, 0.45~0.55mg/L puridoxine hydrochlorides, 0.45~0.55mg/
L nicotinic acid, 99~101mg/L inositols, 0.65~0.75mg/L NAA, 18~22g/L sucrose, 7~8g/L carragheens, pH5.75~
5.85。
Further, step 3) and step 4) in clump bud induction and the condition of culture of shoot proliferation culture be：Temperature 23
~27 DEG C, 9~11hd of illumination-1, 2200~2700Lx of intensity of illumination；Step 5) in the condition of culture of rooting induction culture be：
24~26 DEG C of temperature, 8~10hd of illumination-1, 2200~2700Lx of intensity of illumination.
Further, step 7) described in transplanting medium contain 45~55% yellow mud and 45~55% peat soil matrix.
Further, step 7) described in the specific method of field planting management be：After transplanting first sunshade rate be 90~
95%, relative humidity is 85%~90%, and temperature is culture 7~8 days under conditions of 25~33 DEG C；Then nursery stock is placed in sunshade
Rate is 75%~80%, and humidity is 75%~80%, and temperature is culture 8~20 days under conditions of 25~33 DEG C；Routinely later
Nursery carries out sunshade and water drenching management.
Further, step 7) in transplanting time be 3~June or 9~November.
The beneficial effects of the invention are as follows：
The present invention establish it is a kind of be obtained in that in large quantities by the method for tissue culture of silvery birch clone nursery stock, wherein, this
Invention improves ground by the clump bud inducement cultivation base of silvery birch, proliferated culture medium and root media by studying, and is especially embodied in
Selection to hormone kind, concentration in these three culture mediums, micro and a great number of elements concentration improvement, just ensure that it is silver-colored
The high yield of birch clone nursery stock, growth is fast, and the healthy and strong desired result of nursery stock.
The inventive method acquisition ground is high by the yield of silvery birch clone nursery stock, and the time is fast, and the nursery stock for being obtained is healthy and strong, this
A little nursery stocks are lacked with solving the problems, such as growing industry by silvery birch nursery stock.
The inventive method is to obtain the ground of a large amount of high-quality health by silvery birch by the method for this vegetative propagation of tissue cultures
Nursery stock, the method for this vegetative propagation can ensure that nursery stock is inherited and keeps original maternal plant fine quality of all completely.
Brief description of the drawings
Fig. 1 is the root media containing various concentrations a great number of elements over the ground by the influence of silvery birch tissue culture seedling rooting；
Fig. 2 is the root media containing various concentrations trace element over the ground by the influence of silvery birch tissue culture seedling rooting；
Fig. 3 is different NAA concentration (mg/L) over the ground by the influence of silvery birch tissue culture seedling rooting.
Specific embodiment
A kind ofly by the method for silvery birch tissue cultures, comprise the following steps：
1) collection of explant；
2) sterilization of explant；
3) clump bud Fiber differentiation：Explant after upper step is sterilized is seeded in clump bud inducement cultivation base, and Fiber differentiation 3~
5 months, period need to be forwarded to new clump bud inducement cultivation base 3~5 times, and explant can be induced multiple clump buds；
4) shoot proliferation culture：Upper one-step inducing is gone out into the ground of clump bud by silvery birch tissue culture plant inoculation to subculture multiplication medium
In, often cultivate to be forwarded in new subculture multiplication medium ground by silvery birch propagation seedling in 28~32 days and continue to cultivate；
5) rooting induction culture：By in Multiplying culture highly up to more than 2cm simple bud cut access root media in, training
Support 10~15 days；
6) hardening：Enter the hardening stage after at least 60% propagation seedling starts to take root, the ground of acquisition is taken root by silvery birch
Tissue-cultured seedling is in 3000~6000Lx of light intensity, 26~30 DEG C of temperature condition lower refining seedling 15~20 days；
7) transplant and manage：By in the Nursery stock transplanting after hardening to transplanting medium, field planting management is carried out.
Preferably, step 1) in time of explant collection be June to September, gather 25~30cm parts of new coppice shoot top tip,
And cut off blade.
It is furthermore preferred that step 1) in explant collection time in June to September weather continue sunny 4~6 days after the morning
9 points~10 points.
Preferably, step 2) in the specific method disinfected of explant be：The branch of collection is cleaned up, is cut into
3~4cm is long, with 1~2 stem section of axil, with the HgCl of 0.08~0.12%v/v in gnotobasis2Soaking disinfection 3~
7min, period, constantly shake ensured that thimerosal is fully contacted with branch, then clean with sterile water wash, finally by stem section two ends
0.4~0.6cm is respectively cut, middle stem section containing axil is left as aseptic explant.
Preferably, step 3) in the formula of clump bud inducement cultivation base be：412~413mg/LNH4NO3, 1898~1902mg/
L KNO3, 84.5~85.5mg/L KH2PO4, 369~371mg/L MgSO4·7HO2, 131~133mg/L CaCl2·2HO2、
22~22.5mg/L MnSO4·4HO2, 8.4~8.8mg/L ZnSO4·7HO2, 6~6.4mg/L H3BO3, 0.22~
0.27mg/L Na2MoO4·2HO2, 27.5~28mg/L FeSO4·7HO2, 37~37.5mg/L Na2·EDTA·2HO2、
0.8~0.85mg/L KI, 0.023~0.027mg/L CuSO4·5HO2, 0.023~0.027mg/L CoCl2·6HO2、1.8
~2.2mg/L glycine, 0.08~0.12mg/L thiamine hydrochlorides, 0.48~0.52mg/L puridoxine hydrochlorides, 0.48~
0.52mg/L nicotinic acid, 99~101mg/L inositols, 0.8~1.2g/L PVP, 0.48~0.52mg/L 6-BA, 0.048~
0.052mg/L IBA, 27~32g/L sucrose, 7~8g/L carragheens, pH5.75~5.85.
Preferably, step 3) in clump bud induction condition of culture be：23~27 DEG C of temperature, 9~11hd of illumination-1, illumination
2200~2700Lx of intensity.
Preferably, step 4) in the formula of subculture multiplication medium be：412~413mg/LNH4NO3, 1898~1902mg/
L KNO3, 84.5~85.5mg/L KH2PO4, 369~371mg/L MgSO4·7HO2, 131~133mg/L CaCl2·2HO2、
22~22.5mg/L MnSO4·4HO2, 8.4~8.8mg/L ZnSO4·7HO2, 6~6.4mg/L H3BO3, 0.22~
0.27mg/L Na2MoO4·2HO2, 27.5~28mg/L FeSO4·7HO2, 37~37.5mg/L Na2·EDTA·2HO2、
0.8~0.85mg/L KI, 0.023~0.027mg/L CuSO4·5HO2, 0.023~0.027mg/L CoCl2·6HO2、1.8
~2.2mg/L glycine, 0.08~0.12mg/Lmg/L thiamine hydrochlorides, 0.48~0.52mg/L puridoxine hydrochlorides, 0.48~
0.52mg/L nicotinic acid, 99~101mg/L inositols, 0.8~1.2g/L PVP, 0.2~0.3mg/L 6-BA, 0.048~
0.052mg/L IBA, 27~32g/L sucrose, 7~8g/L carragheens, pH5.75~5.85.
Preferably, step 4) in the condition of culture of subculture Multiplying culture be：23~27 DEG C of temperature, 9~11hd of illumination-1、
2200~2700Lx of intensity of illumination.
Preferably, step 5) in the formula of rooting induction culture medium be：412~413mg/L NH4NO3, 473~477mg/L
KNO3, 42~43mg/L KH2PO4, 92~93mg/L MgSO4·7HO2, 108~112mg/L CaCl2·2HO2, 11~
11.5mg/L MnSO4·4HO2, 4~4.5mg/L ZnSO4·7HO2, 2.8~3.4mg/L H3BO3, 0.12~0.13mg/L
Na2MoO4·2HO2, 13.5~14.5mg/L FeSO4·7HO2, 18.3~18.9mg/L Na2·EDTA·2HO2, 0.41~
0.42mg/L KI, 0.012~0.013mg/L CuSO4·5HO2, 0.012~0.013mg/L CoCl2·6HO2, 1.8~
2.2mg/L glycine, 0.08~0.12mg/L thiamine hydrochlorides, 0.45~0.55mg/L puridoxine hydrochlorides, 0.45~0.55mg/
L nicotinic acid, 99~101mg/L inositols, 0.65~0.75mg/L NAA, 18~22g/L sucrose, 7~8g/L carragheens, pH5.75~
5.85。
Preferably, step 5) in the condition of culture of rooting induction culture be：24~26 DEG C of temperature, 8~10hd of illumination-1、
2200~2700Lx of intensity of illumination.
Preferably, step 7) described in transplanting medium contain 45~55% yellow mud and 45~55% peat soil matrix.
Preferably, step 7) described in the specific method of field planting management be：After transplanting first sunshade rate be 90~
95%, relative humidity is 85%~90%, and temperature is culture 7~8 days under conditions of 25~33 DEG C；Then nursery stock is placed in sunshade
Rate is 75%~80%, and humidity is 75%~80%, and temperature is culture 8~20 days under conditions of 25~33 DEG C；Routinely later
Nursery carries out sunshade and water drenching management.
Preferably, step 7) in transplanting time be 3~June or 9~November.
It is furthermore preferred that step 7) in transplanting time be 3~April.
Preferably, before transplantation, the disinfecting solution of potassium permanganate that medium is with 1/1000 is transplanted, each week uses 1/ after transplanting
800 Bravo and carbendazim is sprayed in turn, prevents miscellaneous bacteria from growing, and starts within 15 days after transplanting fertilising, using 1/1000 it is compound
Fertile solution is sprayed weekly once.
With reference to specific embodiment, the present invention is further illustrated, but is not limited thereto.
The ground of embodiment 1 is by the method for silvery birch tissue cultures
Ground is comprised the following steps that by silvery birch tissue cultures：
(1) collection of explant
In rainfall less June to September, after weather continues sunny 5 days, in 9 points~10 points of the morning, the top tip of new coppice shoot is gathered
25~30cm, cuts off blade and is put into clean freshness protection package, and takes back laboratory in time and carry out disinfection treatment.
(2) sterilization of explant
Branch 30min is soaked with washing powder solution, is then rinsed well in running water down-flow water.Branch is cut into 3~4cm
It is long, with 1~2 stem section of axil, on superclean bench with 0.1% HgCl23~7min of soaking disinfection, period constantly shakes
Dynamic to ensure that thimerosal is fully contacted with branch, then with sterile water wash 4 times, each 5min finally respectively cuts stem section two ends
0.5cm, leaves middle stem section containing axil as aseptic explant, is inoculated into clump bud inducement cultivation base.
(3) clump bud Fiber differentiation
After explant is gathered and sterilized, the induced synthesis clump bud first in clump bud inducement cultivation base, induction time is 3~5
Individual month, midway needed switching and changes fresh culture 3~5 times, the clump bud inductivity about 15% of explant.Sent out through experimental study
Existing, ground needs 6-BA concentration higher during being induced by silvery birch clump bud.Clump bud induction condition of culture be：23~27 DEG C of temperature, light
According to 9~11hd-1, 2200~2700Lx of intensity of illumination.
The formula of above-mentioned clump bud inducement cultivation base is：412.5mg/LNH4NO3、1900mg/L KNO3、85mg/L KH2PO4、
370mg/L MgSO4·7HO2、132mg/L CaCl2·2HO2、22.3mg/L MnSO4·4HO2、8.6mg/L ZnSO4·
7HO2、6.2mg/L H3BO3、0.25mg/LNa2MoO4·2HO2、27.8mg/L FeSO4·7HO2、37.3mg/L Na2·
EDTA·2HO2、0.83mg/L KI、0.025mg/L CuSO4·5HO2、0.025mg/L CoCl2·6HO2, 2mg/L glycine,
0.1mg/L thiamine hydrochlorides, 0.5mg/L puridoxine hydrochlorides, 0.5mg/L nicotinic acid, 100mg/L inositols, 1g/L PVP, 0.5mg/L
6-BA, 0.05mg/L IBA, 30g/L sucrose and 7~8g/L carragheens, pH5.8 (are shown in Table 2).
(4) shoot proliferation culture
The ground that upper one-step inducing goes out clump bud is transferred in subculture multiplication medium by silvery birch tissue-cultured seedling and is bred and expanded numerous.
The condition of culture of shoot proliferation is：Temperature is 25 ± 2 DEG C, illumination 10h/d, and intensity of illumination 2500Lx, switching in every 30 days once, is trained
Support 3~5 months.In order to configure the subculture multiplication medium for compatibly being grown by silvery birch tissue-cultured seedling, the present embodiment is sought to correlation
Foster element is conducted in-depth research.
1. a great number of elements
By the contrast test of a great number of elements, the NH in a great number of elements4NO3、KH2PO4、CaCl2It is influence propagation seedling state
Key factor, NH containing 0.25MS in culture medium4NO3+0.5MS KH2PO4+0.3MS CaCl2·2HO2+1MS KNO3+1MS
MgSO4·7HO2Can make tissue culture breed seedling keep excellent condition, otherwise breed seedling occur top it is withered fall situation.
2. hormone
The concentration that 6-BA is found by studying be determine tissue-cultured seedling proliferation times, height and vitrifying degree it is decisive because
Element.
In order to study the influence that hormone concentration is bred seedling by silvery birch over the ground, the hormone 6-BA concentration in proliferated culture medium is set
4 concentration gradients of 0.2mg/L, 0.3mg/L, 0.4mg/L, 0.5mg/L are counted, 10 bottles per treatment of each concentration, every bottle of inoculation increases
Bud 4 is grown, after cultivating 30 days, proliferation results is counted.
The hormone concentration of table 1 is bred the influence of seedling by silvery birch over the ground
Research shows that the concentration of 6-BA has significantly to proliferation times, the height of propagation seedling, the propagation vitrified degree of seedling
Influence (being shown in Table 1).Although when 6-BA concentration is 0.5mg/L, proliferation times can reach 2 times, and the height for breeding seedling is too low, no
Beneficial to the rooting induction in later stage, and the propagation vitrified ratio of seedling is also higher, and the ratio of healthy seedling can be caused to decline.Comprehensively examine
Consider the factor of each side, the concentration of 6-BA, in 0.2~0.3mg/L, is most suitably by the Multiplying culture of silvery birch tissue-cultured seedling.
In sum, the concentration (table 1) of 6-BA need to be suitably reduced during shoot proliferation culture, optimal shoot proliferation is finally obtained
The formula of culture medium is：Modified MS medium containing 0.2~0.3mg/L 6-BA and 0.05mg/L IBA；
Subculture multiplication medium Ju Ti Pei Fang is：412.5mg/LNH4NO3、1900mg/L KNO3、85mg/L KH2PO4、
370mg/L MgSO4·7HO2、132mg/L CaCl2·2HO2、22.3mg/L MnSO4·4HO2、8.6mg/L ZnSO4·
7HO2、6.2mg/L H3BO3、0.25mg/LNa2MoO4·2HO2、27.8mg/L FeSO4·7HO2、37.3mg/L Na2·
EDTA·2HO2、0.83mg/L KI、0.025mg/L CuSO4·5HO2、0.025mg/L CoCl2·6HO2, 2mg/L glycine,
0.1mg/L thiamine hydrochlorides, 0.5mg/L puridoxine hydrochlorides, 0.5mg/L nicotinic acid, 100mg/L inositols, 1g/L PVP, 0.2~
0.3mg/L 6-BA, 0.05mg/L IBA, 30g/L sucrose, 7~8g/L carragheens, pH5.8 (are shown in Table 2).
(5) rooting induction
When propagation seedling is transferred each time, by the ground in Multiplying culture highly up to more than 2cm by silvery birch tissue culture simple bud
Access rooting induction culture medium is cut, is cultivated 10~15 days, 24~26 DEG C of cultivation temperature, 8~10hd of illumination-1, intensity of illumination
2500lx.In order to configure compatibly by the rooting induction culture medium of silvery birch tissue culture seedling rooting, the present embodiment is to related nutritional unit
Element is conducted in-depth research.
Studied by the content to a great number of elements in root media, trace element, hormone, a great number of elements is worked as in discovery
Content be 0.25MS, micronutrient levels be 0.5MS, NAA concentration be 0.7mg/L when, be best suitable for inductively by silvery birch tissue-cultured seedling
Take root (see Fig. 1, Fig. 2, Fig. 3).
According to the experimental result of Fig. 1~3, be best suitable for be by the prescription of rooting medium of silvery birch tissue culture seedling rooting inductively：
412.5mg/L NH4NO3、475mg/L KNO3、42.5mg/L KH2PO4、92.5mg/L MgSO4·7HO2、110mg/L
CaCl2·2HO2、11.15mg/L MnSO4·4HO2、4.3mg/L ZnSO4·7HO2、3.1mg/L H3BO3、0.125mg/L
Na2MoO4·2HO2、13.9mg/L FeSO4·7HO2、18.65mg/L Na2·EDTA·2HO2、0.415mg/L KI、
0.0125mg/L CuSO4·5HO2、0.0125mg/L CoCl2·6HO2, 2mg/L glycine, 0.1mg/L thiamine hydrochlorides,
0.5mg/L puridoxine hydrochlorides, 0.5mg/L nicotinic acid, 100mg/L inositols, 0.7mg/LNAA, 20g/L sucrose, 7~8g/L carragheens,
PH5.8 (is shown in Table 2).
The ground of table 2 is by the culture medium prescription of silvery birch tissue-culturing rapid propagation
Note：The usage amount of carragheen can be adjusted according to the product of different manufacturers.
(6) hardening
The hardening stage is transferred to by after at least 60% propagation seedling starts to take root.Suitably by silvery birch take root tissue-cultured seedling refining
Slender part is：Intensity of illumination is 6000~8000lx, and 26~30 DEG C of temperature, the suitable hardening time is 15~20 days.By refining
Seedling, up to 4~5cm, well developed root system, nursery stock is sturdy, leaf color jade green for height of seedling of taking root, can with directly transplanting nursery nutrient bag
In.
(7) transplant and manage
The offspring wood that has that hardening will be completed carefully is taken out from blake bottle, and the culture medium of net nursery stock base portion is rinsed with clear water,
The same day is implanted into containing in the nutrient bag that mass percent is 50% yellow mud and 50% peat soil matrix, and nutrient bag specification is 6 ×
11cm。
Ground is 90~95% in sunshade rate by silvery birch tissue-cultured seedling transplant early, and relative humidity is 85%~90%, and temperature is
Cultivated 7~8 days under conditions of 25~33 DEG C；Then it is 75%~80% nursery stock to be placed in into sunshade rate, and humidity is 75%~80%,
Temperature is culture 8~20 days under conditions of 25~33 DEG C；Later routinely nursery carry out sunshade and water drenching management, Nursery stock transplanting into
Motility rate is more than 85%.
Concrete operations are as follows：
1) Nursery stock transplanting the 1st~7 day：Film is added a cover in seedbed, and the shading screen that 1 layer of sunshade rate is 95% is added a cover on film；
Fine day spraying 5~6 times daily, cloudy day spraying 4~5 times daily, the rainy day sprays 2~3 times daily；When temperature is high, on shading screen
Temperature lowering water is drenched, it is 25~33 DEG C to control nursery bed temperature.
2) Nursery stock transplanting the 8th~30 day：The film of two and side sets a ventilating opening at interval of 1 meter on seedbed, in vain
Canopy sunshade rate is 85% sunshade net.During fine day, daily water drenching 4~5 times；Cloudy water drenching 3~4 times daily；If the rainy day, then
Whole day only lid film, daily water drenching 0~1 time.
3) Nursery stock transplanting 31~60 days：Without lid film again, but the sunshade net of lid 50% is still needed on the daytime of fine day, daily
Water drenching 2~3 times；Cloudy day is not required to lid sunshade net, daily water drenching 1~2 time；The small rainy day, lid sunshade net is not required to, is also not required to water drenching；Greatly
Rainy day needs lid film, but need to set ventilating opening at the two and side in seedbed, is not required to lid sunshade net, is also not required to water drenching.
Before Nursery stock transplanting, with 1/1000 disinfecting solution of potassium permanganate, each week uses 1/800 to transplanting medium after transplanting
Bravo and carbendazim spray in turn, prevent miscellaneous bacteria from growing.Start within 15 days after transplanting fertilising, the composite fertilizer using 1/1000 is molten
Liquid is sprayed once for every 1 week.
In the 3~June in Guangdong or 9~11 monthly portables by silvery birch tissue-cultured seedling, wherein 3~April is ground by silvery birch group
The optimal seasonal migration of seedling is trained, and temperature, humidity are suitable for the further culture of nursery stock, reach afforestation height and require.7~8
Month, temperature is too high, and transplanting success is relatively low, is applied fertilizer by intensity, and nursery stock can reach afforestation height.10~November transplants
Also higher, but seedling growth season mistake is survived, spring next year nursery stock is still unable to reach afforestation height.
It is generally artificial culture or natural hybridization product by silvery birch due to originating in Australian ground with application value at present
Kind, most self-sterilities, therefore, ground is difficult to obtain by the seed of silvery birch；Another aspect seed growing is also difficult to keep hybridization new
The good characteristic of kind, after introducing a small amount of maternal plant, only by this pierre technology breeding nursery stock of tissue culture, could promote
Using these new varieties.The inventive method acquisition ground is high by the yield of silvery birch clone nursery stock, and the time is fast, and the nursery stock for being obtained
Stalwartness, these nursery stocks are lacked with solving the problems, such as planting industry by silvery birch nursery stock.
Embodiment 2 is a kind ofly by the method for silvery birch tissue cultures
1) collection of explant：9 points~10 points of the morning after weather continues sunny 6 days in annual June to September, collection is new
25~30cm parts of coppice shoot top tip, and cut off blade；
2) sterilization of explant：The branch of collection is cleaned up, cut into 3~4cm it is long, with 1~2 stem of axil
Section, with the HgCl of 0.08~0.12%v/v in gnotobasis23~7min of soaking disinfection, period, constantly shake ensured thimerosal
It is fully contacted with branch, it is then clean with sterile water wash, stem section two ends are respectively finally cut into 0.4~0.6cm, leave centre and contain
Axil stem section is used as aseptic explant.
3) clump bud Fiber differentiation：Explant after upper step is sterilized is seeded in clump bud inducement cultivation base, Fiber differentiation 4
Month, period need to be forwarded to new clump bud inducement cultivation base 4 times, and a subculture is changed in often culture for one month, and explant can be induced
Go out multiple clump buds；Condition of culture is：25 DEG C of temperature, illumination 10hd-1, intensity of illumination 2500Lx；
The formula of above-mentioned clump bud inducement cultivation base is：412~413mg/L NH4NO3, 1898~1902mg/L KNO3、
84.5~85.5mg/L KH2PO4, 369~371mg/L MgSO4·7HO2, 131~133mg/L CaCl2·2HO2, 22~
22.5mg/L MnSO4·4HO2, 8.4~8.8mg/L ZnSO4·7HO2, 6~6.4mg/L H3BO3, 0.22~0.27mg/L
Na2MoO4·2HO2, 27.5~28mg/L FeSO4·7HO2, 37~37.5mg/L Na2·EDTA·2HO2, 0.8~
0.85mg/L KI, 0.023~0.027mg/L CuSO4·5HO2, 0.023~0.027mg/L CoCl2·6HO2, 1.8~
2.2mg/L glycine, 0.08~0.12mg/L thiamine hydrochlorides, 0.48~0.52mg/L puridoxine hydrochlorides, 0.48~0.52mg/
L nicotinic acid, 99~101mg/L inositols, 0.8~1.2g/L PVP, 0.48~0.52mg/L 6-BA, 0.048~0.052mg/L
IBA, 27~32g/L sucrose, 7~8g/L carragheens, pH5.75~5.85.
4) shoot proliferation culture：Upper one-step inducing is gone out into the ground of clump bud by silvery birch tissue culture plant inoculation to subculture multiplication medium
In, often cultivate to be forwarded in new subculture multiplication medium ground by silvery birch propagation seedling in 28~32 days and continue to cultivate；Condition of culture
For：25 DEG C of temperature, illumination 10hd-1, intensity of illumination 2500Lx；
The formula of above-mentioned subculture multiplication medium is：412~413mg/L NH4NO3, 1898~1902mg/L KNO3、
84.5~85.5mg/L KH2PO4, 369~371mg/L MgSO4·7HO2, 131~133mg/L CaCl2·2HO2, 22~
22.5mg/L MnSO4·4HO2, 8.4~8.8mg/L ZnSO4·7HO2, 6~6.4mg/L H3BO3, 0.22~0.27mg/L
Na2MoO4·2HO2, 27.5~28mg/L FeSO4·7HO2, 37~37.5mg/L Na2·EDTA·2HO2, 0.8~
0.85mg/L KI, 0.023~0.027mg/L CuSO4·5HO2, 0.023~0.027mg/L CoCl2·6HO2, 1.8~
2.2mg/L glycine, 0.08~0.12mg/L thiamine hydrochlorides, 0.48~0.52mg/L puridoxine hydrochlorides, 0.48~0.52mg/
L nicotinic acid, 99~101mg/L inositols, 0.8~1.2g/L PVP, 0.2~0.3mg/L 6-BA, 0.048~0.052mg/L IBA,
27~32g/L sucrose, 7~8g/L carragheens, pH5.75~5.85.
5) rooting induction culture：By in Multiplying culture highly up to more than 2cm simple bud cut access root media in, training
Support 10~15 days；Condition of culture is：245 DEG C of temperature, illumination 9hd-1, intensity of illumination 2700Lx.
The formula of above-mentioned rooting induction culture medium is：412~413mg/LNH4NO3, 473~477mg/L KNO3, 42~
43mg/L KH2PO4, 92~93mg/L MgSO4·7HO2, 108~112mg/L CaCl2·2HO2, 11~11.5mg/L
MnSO4·4HO2, 4~4.5mg/L ZnSO4·7HO2, 2.8~3.4mg/L H3BO3, 0.12~0.13mg/L Na2MoO4·
2HO2, 13.5~14.5mg/L FeSO4·7HO2, 18.3~18.9mg/L Na2·EDTA·2HO2, 0.41~0.42mg/L
KI, 0.012~0.013mg/L CuSO4·5HO2, 0.012~0.013mg/L CoCl2·6HO2, the sweet ammonia of 1.8~2.2mg/L
Acid, 0.08~0.12mg/L thiamine hydrochlorides, 0.45~0.55mg/L puridoxine hydrochlorides, 0.45~0.55mg/L nicotinic acid, 99~
101mg/L inositols, 0.65~0.75mg/L NAA, 18~22g/L sucrose, 7~8g/L carragheens, pH5.75~5.85.
6) hardening：Enter the hardening stage after at least 60% propagation seedling starts to take root, the ground of acquisition is taken root by silvery birch
Tissue-cultured seedling is in 3000~6000Lx of light intensity, 30 DEG C of temperature condition lower refining seedling 15 days；
7) transplant and manage：By the Nursery stock transplanting after hardening to containing 45~55% yellow mud and 45~55% peat soil matrix
Transplanting medium in, carry out field planting management.The time of transplanting is 3~April.Before transplantation, medium is transplanted with 1/1000
Disinfecting solution of potassium permanganate, each week is sprayed in turn with 1/800 Bravo and carbendazim after transplanting, prevents miscellaneous bacteria from growing,
Start within 15 days after transplanting fertilising, sprayed weekly once using 1/1000 composite fertilizer's solution.
The specific method of field planting management is：After transplanting first sunshade rate be 90~95%, relative humidity be 85%~
90%, temperature is culture 7~8 days under conditions of 25~33 DEG C；Then it is 75%~80% nursery stock to be placed in into sunshade rate, and humidity is
75%~80%, temperature is culture 8~20 days under conditions of 25~33 DEG C；Routinely nursery carries out sunshade and spray pipe later
Reason.
Above-described embodiment is the present invention preferably implementation method, but embodiments of the present invention are not by above-described embodiment
Limitation, it is other it is any without departing from Spirit Essence of the invention and the change, modification, replacement made under principle, combine, simplification,
Equivalent substitute mode is should be, is included within protection scope of the present invention.
Claims (7)
1. a kind ofly by the method for silvery birch tissue cultures, it is characterised in that：Comprise the following steps：
1）The collection of explant；The explant is stem section containing axillary bud；
2）The sterilization of explant；
3）Clump bud Fiber differentiation：Explant after upper step is sterilized is seeded in clump bud inducement cultivation base, Fiber differentiation 3~5
Month, period need to be forwarded to new clump bud inducement cultivation base 3~5 times, and explant can be induced multiple clump buds；
4）Shoot proliferation culture：During upper one-step inducing is gone out into the ground of clump bud by silvery birch tissue culture plant inoculation to subculture multiplication medium, often
By silvery birch propagation seedling be forwarded in new subculture multiplication medium on ground in 28~32 days and continue to cultivate by culture；
5）Rooting induction culture：By in shoot proliferation culture highly up to more than 2cm simple bud cut access root media in, training
Support 10~15 days；
6）Hardening：Enter the hardening stage after at least 60% propagation seedling starts to take root, the ground of acquisition is taken root tissue-cultured seedling by silvery birch
In the Lx of light intensity 3000~6000,26~30 DEG C of temperature condition lower refining seedling 15~20 days；
7）Transplanting and management：By in the Nursery stock transplanting after hardening to transplanting medium, field planting management is carried out；
Step 3）The formula of middle clump bud inducement cultivation base is：412~413 mg/L NH4NO3, 1898~1902 mg/L KNO3、
84.5~85.5mg/L KH2PO4、369~371mg/L MgSO4·7H2O、131~133 mg/L CaCl2·2H2O、22~22.5
mg/L MnSO4·4H2O, 8.4~8.8 mg/L ZnSO4·7H2O, 6~6.4 mg/L H3BO3, 0.22~0.27 mg/L
Na2MoO4·2H2O, 27.5~28 mg/L FeSO4·7H2O, 37~37.5 mg/L Na2·EDTA·2H2O, 0.8~
0.85mg/L KI, 0.023~0.027 mg/L CuSO4·5H2O, 0.023~0.027 mg/L CoCl2·6H2O, 1.8~
2.2 mg/L glycine, 0.08~0.12 mg/L thiamine hydrochlorides, 0.48~0.52 mg/L puridoxine hydrochlorides, 0.48~0.52
Mg/L nicotinic acid, 99~101 mg/L inositols, 0.8~1.2 g/L PVP, 0.48~0.52 mg/L 6-BA, 0.048~
0.052mg/L IBA, 27~32g/L sucrose, 7 ~ 8 g/L carragheens, pH5.75~5.85；
Step 4）The formula of middle subculture multiplication medium is：412~413 mg/L NH4NO3, 1898~1902 mg/L KNO3、
84.5~85.5mg/L KH2PO4、369~371mg/L MgSO4·7H2O、131~133 mg/L CaCl2·2H2O、22~22.5
mg/L MnSO4·4H2O, 8.4~8.8 mg/L ZnSO4·7H2O, 6~6.4 mg/L H3BO3, 0.22~0.27 mg/L
Na2MoO4·2H2O, 27.5~28 mg/L FeSO4·7H2O, 37~37.5 mg/L Na2·EDTA·2H2O, 0.8~
0.85mg/L KI, 0.023~0.027 mg/L CuSO4·5H2O, 0.023~0.027 mg/L CoCl2·6H2O, 1.8~
2.2 mg/L glycine, 0.08~0.12 mg/L thiamine hydrochlorides, 0.48~0.52 mg/L puridoxine hydrochlorides, 0.48~
0.52 mg/L nicotinic acid, 99~101 mg/L inositols, 0.8~1.2 g/L PVP, 0.2~0.3 mg/L 6-BA, 0.048~
0.052mg/L IBA, 27~32g/L sucrose, 7 ~ 8 g/L carragheens, pH5.75~5.85；
Step 5）The formula of middle root media is：412~413 mg/L NH4NO3, 473~477 mg/L KNO3, 42~43
mg/L KH2PO4, 92~93 mg/L MgSO4·7H2O, 108~112 mg/L CaCl2·2H2O, 11~11.5 mg/L
MnSO4·4H2O, 4~4.5 mg/L ZnSO4·7H2O, 2.8~3.4mg/L H3BO3, 0.12~0.13 mg/L Na2MoO4·
2H2O, 13.5~14.5 mg/L FeSO4·7H2O, 18.3~18.9mg/L Na2·EDTA·2H2O, 0.41~0.42 mg/L
KI, 0.012~0.013 mg/L CuSO4·5H2O, 0.012~0.013 mg/L CoCl2·6H2O, 1.8~2.2 mg/L are sweet
Propylhomoserin, 0.08~0.12 mg/L thiamine hydrochlorides, 0.45~0.55 mg/L puridoxine hydrochlorides, 0.45~0.55 mg/L cigarettes
Acid, 99~101mg/L inositols, 0.65~0.75 mg/L NAA, 18 ~ 22g/L sucrose, 7 ~ 8g/L carragheens, pH5.75~
5.85。
2. method according to claim 1, it is characterised in that：Step 1）The time of middle explant collection is June to September, is adopted
Collect 25~30cm parts of new coppice shoot top tip, and cut off blade.
3. method according to claim 1, it is characterised in that：Step 2）The specific method that middle explant is disinfected is：
The branch of collection is cleaned up, cut into 3~4cm it is long, with 1~2 stem section of axil, in gnotobasis with 0.08~
The HgCl of 0.12%v/v23~7min of soaking disinfection, period, constantly shake ensured that thimerosal is fully contacted with branch, then with nothing
Bacterium water is cleaned up, and stem section two ends are respectively finally cut into 0.4~0.6cm, leaves middle stem section containing axil as aseptic explant.
4. method according to claim 1, it is characterised in that：Step 3）With step 4）Middle clump bud induction and shoot proliferation training
Foster condition of culture is：23~27 DEG C of temperature, 9~11hd of illumination-1, 2200~2700Lx of intensity of illumination；Step 5）Middle life
The condition of culture of root induction culture is：24~26 DEG C of temperature, 8~10hd of illumination-1, 2200~2700Lx of intensity of illumination.
5. method according to claim 1, it is characterised in that：Step 7）Described in transplanting medium contain 45~55% yellow mud
With 45~55% peat soil matrix.
6. method according to claim 1, it is characterised in that：Step 7）Described in field planting management specific method
For：After transplanting first sunshade rate be 90~95%, relative humidity be 85%~90%, temperature be 25~33 DEG C under conditions of culture 7~
8 days；Then nursery stock is placed in sunshade rate for 75%~80%, humidity is 75%~80%, temperature is to cultivate 8 under conditions of 25~33 DEG C
~20 days；Routinely nursery carries out sunshade and water drenching management later.
7. method according to claim 1, it is characterised in that：Step 7）The time of middle transplanting is 3~June or 9~November.
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