CN105900843B - A kind of method and its culture medium of great bilberry tissue-culturing rapid propagation - Google Patents
A kind of method and its culture medium of great bilberry tissue-culturing rapid propagation Download PDFInfo
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Abstract
The invention discloses a kind of method and its culture medium of great bilberry tissue-culturing rapid propagation.The culture medium provided by the present invention for being used to obtain great bilberry regeneration plant, is consisted of the following composition：NH4NO3、Ca(NO3)2·4H2O、KNO3、KH2PO4、MgSO4·7H2O、MnSO4·4H2O、ZnSO4·7H2O、H3BO3、CuSO4·5H2O、NaMoO4·2H2O, inositol, vitamin B1, nicotinic acid, vitamin B6, glycine, Na2‑EDTA、FeSO4·7H2O, indolebutyric acid, cycocel, sucrose and distilled water.For the thin and delicate phenomenon of great bilberry tissue culture nursery stock, present invention firstly provides cycocel is added into culture medium, coordinate plant hormone, promote the method for great bilberry tissue-cultured seedling rejuvenation, this method can be by strengthening tissue-cultured seedling degree of lignification, strengthen its anti-adversity ability, to improve the survival rate after nursery stock transplanting.Meanwhile method proposes aiding in transplanting with Seedling bag, this method makes the transplanting survival rate of great bilberry tissue-cultured seedling reach 100%.
Description
Technical field
The present invention relates to plant tissue culture technique, the method for more particularly to a kind of great bilberry tissue-culturing rapid propagation and its culture
Base.
Background technology
Great bilberry (Vaccinium uliginosum), also known as bilberry, pasture fruit etc., are a kind of Ericaceaes
(Ericaceae) the edible coloured berry of genus vaccinium (Vaccinium), its fruit is dark blue, delicate mouthfeel, and fruity is pleasant.
Research shows that great bilberry has anti-oxidant, anti-aging, anticancer, prevention of cardiovascular disease, protection eyesight and other effects, has pole
Good health-care efficacy.In China, great bilberry integrated distribution is in the big Xiaoxinanlin Mountains and Changbaishan area, wherein Daxinganling District
For its maximum natural distributed area.Investigation display, the natural distributed area of Daxinganling District great bilberry have 150,000 hectares with
On, year about 20,000 tons of reserves.But under the driving of interests, the wild great bilberry in China is developed by damage type.People
Class is often destructive in a manner of rolling over branch strip leaf to harvest great bilberry fruit, and irreversible damage is caused to its natural habitat
Wound.It is reported that the phenomenon that Resource Degradation has prematurely occurred in the wild great bilberry in China, population area reduces, great bilberry
Endangered plants monoid can be included in.Therefore, it is necessary to take measures, great bilberry protection of resources system is established.Wherein, can implement
Property strong, quick safeguard measure be exactly to establish great bilberry resource to return plant project, i.e. artificial culture great bilberry nursery stock, and will
The accordingly original area that its Hui Zhi is destroyed to resource, to ensure the sustainability of development of resources, conserve forests ecologic structure.
At present, studies have reported that the foundation of great bilberry regenerating system, existing system can realize great bilberry again
It is raw.But because great bilberry tissue-cultured seedling stalk is generally very thin, and it is more difficult to be taken root in its bottle, taking root, time-consuming (is usually
40-90 days), and root is thinner and more delicate.Meanwhile great bilberry tissue culture transplantation of seedlings correlative study is more short of, survival rate is relatively low.The present situation
It is invisible to add great bilberry seedling cost, the process of great bilberry industrial seedling rearing is significantly limit, bog bilberry is constrained and gets over
Tangerine protection of resources effect.
The content of the invention
In order to make up the deficiency in above field, the invention provides a kind of method of great bilberry tissue-culturing rapid propagation and its culture
Base.
The culture medium provided by the present invention for being used to obtain great bilberry regeneration plant, is consisted of the following composition：
NH4NO3、Ca(NO3)2·4H2O、KNO3、KH2PO4、MgSO4·7H2O、MnSO4·4H2O、ZnSO4·7H2O、
H3BO3、CuSO4·5H2O、NaMoO4·2H2O, inositol, vitamin B1, nicotinic acid, vitamin B6, glycine, Na2-EDTA、
FeSO4·7H2O, indolebutyric acid, cycocel, sucrose and distilled water；
Above composition concentration in the culture medium is respectively：
NH4NO3 0.4g/L、Ca(NO3)2·4H2O 0.684g/L、KNO3 0.19g/L、KH2PO4 0.17g/L、
MgSO4·7H2O 0.37g/L、MnSO4·4H2O 22.3mg/L、ZnSO4·7H2O 8.6mg/L、H3BO3 6.2mg/L、
CuSO4·5H2O 0.25mg/L、NaMoO4·2H2O 0.25mg/L, inositol 100mg/L, vitamin B1 0.1mg/L, nicotinic acid
0.5mg/L, vitamin B6 0.5mg/L, glycine 2.0mg/L, Na2-EDTA 37.3mg/L、FeSO4·7H2O 27.8mg/L、
Indolebutyric acid 0.1mg/L-0.5mg/L, cycocel 0mg/L-10mg/L and sucrose 20g/L.
The culture medium for being used to obtain great bilberry regeneration plant consists of the following composition：
NH4NO3、Ca(NO3)2·4H2O、KNO3、KH2PO4、MgSO4·7H2O、MnSO4·4H2O、ZnSO4·7H2O、
H3BO3、CuSO4·5H2O、NaMoO4·2H2O, inositol, vitamin B1, nicotinic acid, vitamin B6, glycine, Na2-EDTA、
FeSO4·7H2O, indolebutyric acid, cycocel, sucrose and distilled water；
Above composition it is described be used to obtain the culture medium of great bilberry regeneration plant in concentration be respectively：
NH4NO3 0.4g/L、Ca(NO3)2·4H2O 0.684g/L、KNO3 0.19g/L、KH2PO4 0.17g/L、
MgSO4·7H2O 0.37g/L、MnSO4·4H2O 22.3mg/L、ZnSO4·7H2O 8.6mg/L、H3BO3 6.2mg/L、
CuSO4·5H2O 0.25mg/L、NaMoO4·2H2O 0.25mg/L, inositol 100mg/L, vitamin B1 0.1mg/L, nicotinic acid
0.5mg/L, vitamin B6 0.5mg/L, glycine 2.0mg/L, Na2-EDTA 37.3mg/L、FeSO4·7H2O 27.8mg/L、
Indolebutyric acid 0.5mg/L, cycocel 5mg/L and sucrose 20g/L.
It is by coagulator and institute present invention also offers a kind of solid medium for being used to obtain great bilberry regeneration plant
State the solid medium being made into for obtaining the culture medium of great bilberry regeneration plant.
Concentration of the coagulator in the solid medium is 9g/L；
The coagulator is agar.
The pH value of the culture medium is 5.4.
Present invention also offers a kind of method for obtaining great bilberry regeneration plant, comprise the following steps：
Great bilberry regrowth is inoculated into and described is used to obtain to carry out on the solid medium of great bilberry regeneration plant
Rooting induction, that is, obtain great bilberry regeneration plant.
Methods described also includes the great bilberry Transplantation of Regenerated Plantlets using Seedling bag to being equipped with cultivation matrix seedling basin
The step of auxiliary transplanting.
The Seedling bag is that top carries self-sealed article, transparent, the rigid plastics pocket of lower openings, its underpart opening and seedling
Basin caliber size is consistent；
Described with Seedling bag auxiliary transplanting is to entangle seedling basin with Seedling bag, the self-sealed article on sealing Seedling bag top, and uses rubber
Rubber band ties Seedling bag bottom, ensures the sealing of the planting environment；
The cultivation matrix is that volume ratio is 3:1 turfy soil：Vermiculite.
The great bilberry regrowth is obtained by the method comprised the following steps：
Broken up with induction culture medium induction great bilberry aseptic seedling, culture obtains great bilberry regrowth；
The induction culture medium consists of the following composition：
NH4NO3、Ca(NO3)2·4H2O、KNO3、KH2PO4、MgSO4·7H2O、MnSO4·4H2O、ZnSO4·7H2O、
H3BO3、CuSO4·5H2O、NaMoO4·2H2O, inositol, vitamin B1, nicotinic acid, vitamin B6, glycine, Na2-EDTA、
FeSO4·7H2O, zeatin, indolebutyric acid, sucrose and distilled water；
Above composition concentration in the culture medium is respectively：
NH4NO3 0.4g/L、Ca(NO3)2·4H2O 0.684g/L、KNO3 0.19g/L、KH2PO4 0.17g/L、
MgSO4·7H2O 0.37g/L、MnSO4·4H2O 22.3mg/L、ZnSO4·7H2O 8.6mg/L、H3BO3 6.2mg/L、
CuSO4·5H2O 0.25mg/L、NaMoO4·2H2O 0.25mg/L, inositol 100mg/L, vitamin B1 0.1mg/L, nicotinic acid
0.5mg/L, vitamin B6 0.5mg/L, glycine 2.0mg/L, Na2-EDTA 37.3mg/L、FeSO4·7H2O 27.8mg/L、
Zeatin 1.0mg/L-2.5mg/L, indolebutyric acid 0mg/L-0.5mg/L and sucrose 20g/L.
The induction culture medium consists of the following composition：
NH4NO3、Ca(NO3)2·4H2O、KNO3、KH2PO4、MgSO4·7H2O、MnSO4·4H2O、ZnSO4·7H2O、
H3BO3、CuSO4·5H2O、NaMoO4·2H2O, inositol, vitamin B1, nicotinic acid, vitamin B6, glycine, Na2-EDTA,
FeSO4·7H2O, zeatin, indolebutyric acid, sucrose and distilled water；
Above composition concentration in the culture medium is respectively：
NH4NO3 0.4g/L、Ca(NO3)2·4H2O 0.684g/L、KNO3 0.19g/L、KH2PO4 0.17g/L、
MgSO4·7H2O 0.37g/L、MnSO4·4H2O 22.3mg/L、ZnSO4·7H2O 8.6mg/L、H3BO3 6.2mg/L、
CuSO4·5H2O 0.25mg/L、NaMoO4·2H2O 0.25mg/L, inositol 100mg/L, vitamin B1 0.1mg/L, nicotinic acid
0.5mg/L, vitamin B6 0.5mg/L, glycine 2.0mg/L, Na2-EDTA 37.3mg/L、FeSO4·7H2O 27.8mg/L、
Zeatin 1.0mg/L, indolebutyric acid 0.1mg/L and sucrose 20g/L.
Usual cycocel can impose limitation crop overgrowing outside, there is the effect of promotion is beared fruit.It is thin and delicate for great bilberry nursery stock
Phenomenon, present invention firstly provides cycocel is added into culture medium, coordinate plant hormone, promote great bilberry tissue-cultured seedling rejuvenation
Method, this method can strengthen its anti-adversity ability, to improve surviving after nursery stock transplanting by strengthening tissue-cultured seedling degree of lignification
Rate.
Further, since great bilberry tissue culture transplantation of seedlings initial stage, its root system and branches and leaves still in recover, the laundering period, its is external
The phylactic power defensive power on boundary is weaker, and particularly water holding capacity is poor.Therefore, it is necessary to provide air humidity is higher, air flow compared with
Few stable environment is to ensure great bilberry tissue-cultured seedling transplant survival.Between above-mentioned background, the present invention propose a kind of low cost,
The making and use (Fig. 3) of reusable and easy to operate Seedling bag.The Seedling bag can ensure great bilberry tissue-cultured seedling
Transplant environment air-flow and humidity stabilization, coordinate taken root in great bilberry bottle proposed by the present invention, strong sprout method, bog bilberry can be made
Blueberry tissue-cultured seedling transplanting survival rate is up to 100%.
Brief description of the drawings
Fig. 1 is induction situation of the different culture media to great bilberry tissue-cultured seedling.
Fig. 2 is rooting induction situation of the different culture media to great bilberry tissue-cultured seedling；Wherein A is control；B is addition IBA
0.1mg/L；C is addition IBA 0.5mg/L；D is addition IBA 0.5mg/L+ cycocels 5mg/L.
Fig. 3 is Seedling bag proposed by the present invention and its nursery effect；Wherein, A is with Seedling bag button basin culture after transplanting；B
For culture 2 months after growth conditions.
Fig. 4 is the growing state after great bilberry tissue culture transplantation of seedlings 2 months；Wherein, A is not carry out i.e. overlay film of being taken root in bottle
Transplanting；B is Raising strong seedli after being taken root in bottle；C is to aid in transplanting with Seedling bag after taking root in bottle.
Embodiment
Instrument and reagent：Superclean bench (Beijing Dong Lianhaer), IBA (indolebutyric acid, Beta-indolebutyric
Acid), ZT (zeatin, Zeatin), 2,4D (2,4-Dichlorophenoxyacetic acid), TDZ (Thidiazuron,
Thidiazuron), NAA (methyl α-naphthyl acetate, 1-Naphthaleneacetic acid), 6BA (6-Benzylaminopurine), short
Strong element, agar powder, sucrose are purchased from Beijing Co., Ltd of Kechuang Chendu XinDa, and remaining reagent do not indicated especially is all purchased from Beijing
Chemical company.
Experiment material：Great bilberry aseptic seedling.Wild great bilberry picks up from Daxinganling District, with working as its strong branch
Year, raw tender stem was explant, by tissue culture technique, induction axillary bud regeneration, obtains sterile tissue-cultured seedling and is used for subsequent experimental.
The aseptic seedling can obtain from Institute of Botany, Chinese Academy of Sciences's flowers physiology with genetic breeding research group.Recorded great bilberry
The non-patent literature of (Daxing'an Mountainrange area wild species) is：Care for relation by marriage, He Shanan, what rainbow, Wang Chuanyong, Wu Wenlong (2001) blue berry with
Cranberry (the 1st edition) Beijing：Chinese agriculture publishing house .pp.25-33,142-187.
The efficient tissue-culturing rapid propagation of embodiment 1, great bilberry and the method for transplanting
First, great bilberry inductive differentiation medium and root media are prepared
1st, improvement WPM is prepared0Basal medium：
It is female to measure 50mL improvement WPM a great number of elements mother liquor, 5mL improvement WPM trace elements mother liquor, 5mL improvement WPM organic matters
Liquid, 5mL improvement WPM mother liquid of iron salt, 20mg sucrose, are mixed, with sulfuric acid or phosphorus acid for adjusting pH to 5.4, and constant volume final volume is extremely
1000ml；9g agar is added, 121 DEG C of high-temperature sterilization 20min, is cooled to 65 DEG C or so, it is standby.
The improvement WPM mother liquors formula that the present embodiment is prepared is as shown in table 1：
The improvement WPM mother liquor formulas that the present embodiment of table 1 is prepared
Remarks：All reagents are all placed in 4 DEG C of refrigerators and preserved after preparing.
2nd, it is formulated for the hormone of great bilberry induction and rooting induction
Weigh respectively 10mg ZT (zeatin, Zeatin), IBA (indolebutyric acid, Beta-indolebutyric acid),
2,4D (2,4-Dichlorophenoxyacetic acid), TDZ (Thidiazuron, Thidiazuron), NAA (methyl α-naphthyl acetate, 1-
Naphthaleneacetic acid), 6BA (6-Benzylaminopurine) powder, and be settled to respectively with straight alcohol
10ml；10mg cycocel powder is weighed, 10ml is settled to distilled water；By above-mentioned hormone in super-clean bench, with 0.22 μm of filter
Filtration sterilization.Hormone after sterilizing is placed in 4 DEG C of storages, standby.Hereafter all operations are carried out in super-clean bench, used tool
It is 121 DEG C, the article after the 20min that sterilizes.
3rd, the culture medium for great bilberry induction is prepared
(1) sterilized basal medium is improved into WPM in super-clean bench0Packing is into tissue culture bottle after mixing, and every bottle about
50ml；
(2) according to table 2, corresponding hormone is added into the culture medium dispensed, after mixing, is stood, is used after its solidification；
Every kind of culture medium prepares at least 3 bottles；
The induction culture medium prescription that the present embodiment of table 2 is prepared
Improve WPM0Basal medium consists of the following composition：
NH4NO3、Ca(NO3)2·4H2O、KNO3、KH2PO4、MgSO4·7H2O、MnSO4·4H2O、ZnSO4·7H2O、
H3BO3、CuSO4·5H2O、NaMoO4·2H2O, inositol, vitamin B1, nicotinic acid, vitamin B6, glycine, Na2-EDTA、
FeSO4·7H2O；
Above composition is in improvement WPM0Concentration is respectively in basal medium：
NH4NO3 0.4g/L、Ca(NO3)2·4H2O 0.684g/L、KNO3 0.19g/L、KH2PO4 0.17g/L、
MgSO4·7H2O 0.37g/L、MnSO4·4H2O 22.3mg/L、ZnSO4·7H2O 8.6mg/L、H3BO3 6.2mg/L、
CuSO4·5H2O 0.25mg/L、NaMoO4·2H2O 0.25mg/L, inositol 100mg/L, vitamin B1 0.1mg/L, nicotinic acid
0.5mg/L, vitamin B6 0.5mg/L, glycine 2.0mg/L, Na2-EDTA 37.3mg/L、FeSO4·7H2O 27.8mg/L。
4th, the culture medium for great bilberry rooting induction is prepared
(1) sterilized basal medium is improved into WPM in super-clean bench0Packing is into tissue culture bottle after mixing, and every bottle about
50ml；
(2) according to table 3, corresponding hormone is added into the culture medium dispensed, after mixing, is stood, is used after its solidification；
Every kind of culture medium prepares at least 3 bottles；
The rooting induction culture medium prescription that the present embodiment of table 3 is prepared
Improve WPM0Basal medium consists of the following composition：
NH4NO3、Ca(NO3)2·4H2O、KNO3、KH2PO4、MgSO4·7H2O、MnSO4·4H2O、ZnSO4·7H2O、
H3BO3、CuSO4·5H2O、NaMoO4·2H2O, inositol, vitamin B1, nicotinic acid, vitamin B6, glycine, Na2-EDTA、
FeSO4·7H2O；
Above composition is in improvement WPM0Concentration is respectively in basal medium：
NH4NO3 0.4g/L、Ca(NO3)2·4H2O 0.684g/L、KNO3 0.19g/L、KH2PO4 0.17g/L、
MgSO4·7H2O 0.37g/L、MnSO4·4H2O 22.3mg/L、ZnSO4·7H2O 8.6mg/L、H3BO3 6.2mg/L、
CuSO4·5H2O 0.25mg/L、NaMoO4·2H2O 0.25mg/L, inositol 100mg/L, vitamin B1 0.1mg/L, nicotinic acid
0.5mg/L, vitamin B6 0.5mg/L, glycine 2.0mg/L, Na2-EDTA 37.3mg/L、FeSO4·7H2O 27.8mg/L。
2nd, the induction of great bilberry
(1) after the cooling of induction culture medium, solidification, great bilberry aseptic seedling is cut, the bog bilberry for being respectively connected to table 2 is got over
Tangerine induction culture medium；
(2) tissue-cultured seedling is positioned over 20 (± 5) DEG C, 30-40d, induction is cultivated under the conditions of 1200lx, periodicity of illumination 16h/d
It breaks up, and obtains great bilberry regeneration tissue-cultured seedling.
3rd, the culture of rootage of great bilberry tissue-cultured seedling
(1) highly about 3-4cm great bilberry regeneration tissue-cultured seedling is cut with sharp scissors, is inserted vertically into great bilberry
In rooting induction culture medium, every bottle is accessed 30 plants；
(2) tissue culture bottle after inoculation is placed in 20 (± 5) DEG C, 30-40d is cultivated under the conditions of 1200lx, periodicity of illumination 16h/d
Statistics and analyze data afterwards.
4th, the transplanting of great bilberry rooted seedling
(1) the great bilberry tissue-cultured seedling taken root is taken out, the culture medium that its root is cleaned with flowing water (pays attention to the cleaning step
Light and slow operation is needed, is completely cured with disposable)；
(2) it is turfy soil by clean great bilberry transplantation of seedlings to cultivation matrix of taking root：Vermiculite=3：(carried in 1 seedling basin
It is preceding cultivation matrix is poured it is permeable), by table 4 carry out corresponding overlay film or set Seedling bag processing, it is every kind of processing comprise at least 100 plants
Seedling；Nursery stock after field planting is placed in 20 (± 5) DEG C, counts and divides after 30-40d are cultivated under the conditions of 1200lx, periodicity of illumination 16h/d
Analyse data.
Method is managed and protected in the transplanting that the present embodiment of table 4 is carried out
5th, result
(1) because great bilberry blade is thin and small, it is easy to browning, dried up in operation, therefore compared to leaf
Piece, the tender stem of great bilberry are more suitable for being used as explant, evoking adventive bud regeneration.Experimental result (Fig. 1, table 5) shows, for sincere
The induction of this blueberry, TDZ and zeatin (ZT) play an important roll, and 2,4D is invalid.To the training containing 2.0mg/L TDZ
Great bilberry differentiation can be effectively facilitated by supporting the NAA or IBA of addition low concentration in base.Present invention demonstrates that simultaneously added with TDZ and IBA
Culture medium DSF1 culture can be promoted efficiently to break up, wherein the incision of great bilberry explant and any connect with culture medium
Tactile tip segment has the callus generation for spreading all over red bud point, and has also germinated a large amount of regeneration at the axillary bud of explant
Bud.The culture medium DSF2 added with TDZ and NAA also can induce a large amount of callus and generate simultaneously, but the callus is only being cut mostly
Produced at mouthful, differentiation and regeneration effect is not as good as DSF1.But all there is slow-growing, elongation in the regrowth in DSF1 and DSF2 culture mediums
The problem of difficult, it is necessary to change the subculture medium that nursery stock can be promoted to extend in time.And have for plants such as matrimony vines and preferably divide
2, the 4D for changing inducing effect is not suitable for great bilberry then, and the explant being connected on the culture medium can not break up generation regeneration bud,
Also axillary bud, most gradually agings, withered death can not be germinated.
The great bilberry induction result of table 5
ZT has efficient differentiation-inducing action, is commonly used for the induction of the plant of difficult differentiation.The study show that with
ZT concentration from 0.5-2.5mg/L to change, the differentiation rate of great bilberry also increases therewith.Low concentration zeatin (≤
It can 0.5mg/L) promote explant axillary bud sprouting well, and it extends speed, explant can with culture medium contact position
Form a small amount of callus；The zeatin (1-2.5mg/L) of higher concentration can stimulate explant to break up at a high level, whole in ZT
Concentration is that Extending culture time, the differentiation rate of great bilberry can surpass 100 in 2.5mg/L culture medium.But with differentiation water
Flat raising, vitrified probability, which occurs, in differentiation seedling also to be increased, and high concentration zeatin breaks up obtained tissue-cultured seedling stalk mistake
In very thin, transplant survival is not easy.Therefore, 1.0mg/L zeatin is the optium concentration for inducing great bilberry differentiation, and thereto
The IBA (0.1-0.5mg/L) of addition low concentration has certain strong sprout effect, can reduce the very thin degree of nursery stock, but the IBA of high concentration
Explant differentiation and elongation can be suppressed.In addition, compared with the culture medium added with TDZ, bog bilberry can be induced added with ZT culture medium
Blueberry, which is efficiently broken up, produces a large amount of regeneration buds, and the regeneration bud can rapid elongation simultaneously.Therefore can be added with ZT culture medium simultaneously
To squamous subculture, the complicated degree that culture medium prepares work is reduced, shortens the great bilberry tissue culture cycle.To sum up, it is added with
ZT 1.0mg/L and IBA 0.1mg/L improvement WPM0Culture medium (i.e. culture medium DSF9) is the optimal induction training of great bilberry
Support base.
(2) IBA (0.1-0.5mg/L) of low concentration can effectively facilitate great bilberry tissue-cultured seedling and taken root in bottle, high
The IBA (1.0-2.0mg/L) of concentration is taken root to it with obvious inhibiting effect, and IBA concentration is higher, and inhibitory action is more obvious
(Fig. 2, table 6).Further, since great bilberry tissue-cultured seedling is typically more very thin, degeneration-resistant level is poor, during to increase its transplanting into
Motility rate, this research with the addition of the cycocel of doses in its root media, aid in strong sprout, reduce nursery stock overgrowing, promote
Its stalk is sturdy.As a result show when IBA concentration levels are relatively low (0.1mg/L), while add cycocel and can significantly inhibit and take root,
Rooting rate is set to reduce more than 50%；When IBA concentration levels are higher (1.0mg/L), while add the inhibition of cycocel
Then will not be excessively obvious；And at IBA levels moderate (0.5mg/L), the cycocel for adding low dosage can be with hestening rooting, and energy
Enough effectively facilitate the increase of tissue-cultured seedling degree of lignification.Therefore this research filters out improvement WPM0Basal medium adds IBA
0.5mg/L+ cycocels 5mg/L is its optimal rooting induction culture medium (SG8), and rooting rate is 81.47 ± 3.48%.
The great bilberry tissue-cultured seedling rooting induction result of table 6
(3) this research is not to carry out the transplant survival taken root in bottle, the great bilberry tissue-cultured seedling of strong sprout is directly transplanted
Rate contrasts Raising strong seedli and the transplanting effect for aiding in transplanting with Seedling bag (Fig. 3) after being taken root in bottle respectively as control.Transplanting 30
Survival rate is counted after it, as a result (Fig. 4) shows, does not carry out taking root in bottle, the great bilberry tissue-cultured seedling of strong sprout its transplanting survival rate
Up to 69.37 ± 4.95%, the tissue culture shoot survival percent of Raising strong seedli is 73.79 ± 3.00% after being taken root in bottle, and uses Seedling bag
The plant percent of transplanting is aided in up to 100%.
It is worth noting that, seedling growth situation is observed in transplanting again after 60 days, find not carry out the tissue culture taken root in bottle
Its survival rate of seedling drops to 54.89 ± 4.01%, and the data than after transplanting 1 each moon have dropped nearly 15%, and plant height changes
Seldom, almost do not increase, but the tissue-cultured seedling after being taken root in bottle shows as stable grow.Meanwhile that originally researchs and proposes is used to aid in
The Seedling bag of transplanting can largely maintain the stability of tissue cultured seedling growth environment, including air humidity, temperature and sky
Flow of air, this gradually adapts to extraneous growing environment after being advantageous to tissue-cultured seedling bottle outlet, there is provided preferable spatial transition, greatly
Improve plant percent, and plant strain growth speed.In this research, 150 have been transplanted with Seedling bag auxiliary method for transplanting
Remaining strain great bilberry tissue-cultured seedling, has been survived, and come along fine.
As a result show, great bilberry differential medium proposed by the present invention can induce tissue-cultured seedling efficiently to break up, and this hair
Rooting method is promoting great bilberry bottle in the great bilberry tissue-cultured seedling bottle for adding cycocel and IBA in the medium of bright proposition
While inside taking root (rooting rate is up to more than 80%), it is possible to achieve strong sprout, greatly improve the lignifying of great bilberry tissue-cultured seedling
Degree, anti-adversity ability during great bilberry tissue culture transplantation of seedlings can be effectively increased, and this method is simple to operate, application is strong.Together
When, the inventive method proposes the method that Seedling bag aids in tissue culture transplantation of seedlings, and this method can realize great bilberry tissue-cultured seedling
100% transplanting survival rate, if being used for industrial seedling rearing, enterprise's planting percent can be greatly improved, entreprise cost is reduced, has had
Application value.It is easy to operate meanwhile Seedling bag design is simple, and can reuse.In addition, the Seedling bag is applied to respectively
The seedling basin utensil of kind shape, can be used for the cultivation of other rare materials, has preferable promotional value.Generally, originally
The transplantation of seedlings cycle that goes out of great bilberry tissue-cultured seedling is foreshortened to 30-40d (being counted from culture of rootage) by invention from more than 60d, and
Strong sprout process is completed while inducing tissue culture seedling rooting, with the use of Seedling bag so that great bilberry tissue-cultured seedling transplanting survival rate
Improve to 100%, so, this method for great bilberry industrial seedling rearing and other tissue-cultured seedling wood it is thin and delicate, take root difficult and move
The cultivation for planting the plants such as difficult work is significant.
Claims (3)
1. a kind of method for obtaining Daxing'an Mountainrange great bilberry regeneration plant, comprises the following steps：
Daxing'an Mountainrange great bilberry tender stem is inoculated on induction culture medium, culture obtains Daxing'an Mountainrange great bilberry regeneration
Tissue-cultured seedling, then Daxing'an Mountainrange great bilberry regrowth is seeded to rooting induction culture medium and carries out rooting induction, that is, gone in in a big way
Pacify ridge great bilberry regeneration plant；The Daxing'an Mountainrange great bilberry Transplantation of Regenerated Plantlets is extremely equipped with cultivation matrix seedling basin again
In, aid in transplanting with Seedling bag；The Seedling bag is that top carries self-sealed article, transparent, the rigid plastics pocket of lower openings, its
Lower openings are consistent with seedling basin caliber size；Described with Seedling bag auxiliary transplanting is to entangle seedling basin with Seedling bag, seals Seedling bag
The self-sealed article on top, and Seedling bag bottom is tied with rubber band, ensure the sealing of the planting environment；The cultivation matrix is body
Product is than being 3:1 turfy soil：Vermiculite；
The induction culture medium consists of the following composition：
NH4NO3、Ca(NO3)2·4H2O、KNO3、KH2PO4、MgSO4·7H2O、MnSO4·4H2O、ZnSO4·7H2O、H3BO3、
CuSO4·5H2O、NaMoO4·2H2O, inositol, vitamin B1, nicotinic acid, vitamin B6, glycine, Na2-EDTA、FeSO4·
7H2O, zeatin, indolebutyric acid, sucrose and distilled water；
Above composition concentration in the culture medium is respectively：
NH4NO3 0.4 g/L、Ca(NO3)2·4H2O 0.684 g/L、KNO3 0.19 g/L、KH2PO4 0.17 g/L、MgSO4·
7H2O 0.37 g/L、MnSO4·4H2O 22.3 mg/L、ZnSO4·7H2O 8.6 mg/L、H3BO3 6.2 mg/L、CuSO4·
5H2O 0.25 mg/L、NaMoO4·2H2The mg/L of O 0.25, the mg/L of inositol 100, the mg/L of vitamin B1 0.1, nicotinic acid 0.5
Mg/L, the mg/L of vitamin B6 0.5, glycine 2.0 mg/L, Na2-EDTA 37.3 mg/L、FeSO4·7H2O 27.8 mg/
L, the mg/L -2.5 mg/L of zeatin 1.0, mg/L -0.5 mg/L of indolebutyric acid 0 and the g/L of sucrose 20；
The rooting induction culture medium consists of the following composition：
NH4NO3、Ca(NO3)2·4H2O、KNO3、KH2PO4、MgSO4·7H2O、MnSO4·4H2O、ZnSO4·7H2O、H3BO3、
CuSO4·5H2O、NaMoO4·2H2O, inositol, vitamin B1, nicotinic acid, vitamin B6, glycine, Na2-EDTA、FeSO4·
7H2O, IBA, cycocel, agar and water；
Above composition concentration in rooting induction culture medium is respectively：
NH4NO3 0.4 g/L、Ca(NO3)2·4H2O 0.684 g/L、KNO3 0.19 g/L、KH2PO4 0.17 g/L、MgSO4·
7H2O 0.37 g/L、MnSO4·4H2O 22.3 mg/L、ZnSO4·7H2O 8.6 mg/L、H3BO3 6.2 mg/L、CuSO4·
5H2O 0.25 mg/L、NaMoO4·2H2The mg/L of O 0.25, the mg/L of inositol 100, the mg/L of vitamin B1 0.1, nicotinic acid 0.5
Mg/L, the mg/L of vitamin B6 0.5, glycine 2.0 mg/L, Na2-EDTA 37.3 mg/L、FeSO4·7H2O 27.8 mg/
L, IBA 0.1 mg/L -1 mg/L, the mg/L -10mg/L of cycocel 5 and agar 9g/L.
A kind of 2. method for obtaining Daxing'an Mountainrange great bilberry regeneration plant according to claim 1, it is characterised in that：
The induction culture medium consists of the following composition：
NH4NO3、Ca(NO3)2·4H2O、KNO3、KH2PO4、MgSO4·7H2O、MnSO4·4H2O、ZnSO4·7H2O、H3BO3、
CuSO4·5H2O、NaMoO4·2H2O, inositol, vitamin B1, nicotinic acid, vitamin B6, glycine, Na2-EDTA、FeSO4·
7H2O, zeatin, indolebutyric acid, sucrose and distilled water；
Above composition concentration in the culture medium is respectively：
NH4NO3 0.4 g/L、Ca(NO3)2·4H2O 0.684 g/L、KNO3 0.19 g/L、KH2PO4 0.17 g/L、MgSO4·
7H2O 0.37 g/L、MnSO4·4H2O 22.3 mg/L、ZnSO4·7H2O 8.6 mg/L、H3BO3 6.2 mg/L、CuSO4·
5H2O 0.25 mg/L、NaMoO4·2H2The mg/L of O 0.25, the mg/L of inositol 100, the mg/L of vitamin B1 0.1, nicotinic acid 0.5
Mg/L, the mg/L of vitamin B6 0.5, glycine 2.0 mg/L, Na2-EDTA 37.3 mg/L、FeSO4·7H2O 27.8 mg/
L, the mg/L of zeatin 1.0, the mg/L of indolebutyric acid 0.1 and the g/L of sucrose 20.
3. a kind of method for obtaining Daxing'an Mountainrange great bilberry regeneration plant according to claim 1 or 2, its feature exist
In the condition of the induction culture is that temperature is 20（±5）DEG C, light intensity 1200Lux, the photoperiod be 16h/d under cultivate 30
My god -40 days；
The condition of the culture of rootage is that temperature is 20（±5）DEG C, light intensity 1200Lux, the photoperiod be 16h/d under cultivate 30 days-
40 days.
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