CN105900843A - Bog bilberry tissue culture fast breeding method and culture medium thereof - Google Patents
Bog bilberry tissue culture fast breeding method and culture medium thereof Download PDFInfo
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- CN105900843A CN105900843A CN201610306073.2A CN201610306073A CN105900843A CN 105900843 A CN105900843 A CN 105900843A CN 201610306073 A CN201610306073 A CN 201610306073A CN 105900843 A CN105900843 A CN 105900843A
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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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- 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
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Abstract
The invention discloses a bog bilberry tissue culture fast breeding method and a culture medium thereof. The culture medium for obtaining bog bilberry regeneration plants provided by the invention is prepared from the following components including NH4NO3, Ca(NO3)2.4H2O, KNO3, KH2PO4, MgSO4.7H2O, MnSO4.4H2O, ZnSO4.7H2O, H3BO3, CuSO4.5H2O, NaMoO4.2H2O, phaseomannite, vitaminB1, nicotinic acid, vitaminB6, glycine, Na2-EDTA, FeSO4.7H2O, hormodin, cycocel, sucrose and distilled water. By aiming at the thin and weak phenomena of bog bilberry tissue culture seedlings, the invention provides a method of adding the cycocel into the culture medium to promote the rejuvenation of the bog bilberry tissue culture seedlings in a way of being matched with plant hormone for the first time; the method has the advantages that the adversity ability of the tissue culture seedlings can be enhanced through enhancing the tissue culture seedling lignifications degree, so that the survival rate after the seedling transplanting is improved. Meanwhile, the method provides the auxiliary transplanting by a seedling culture bag. By using the method, the transplanting survival rate of the bog bilberry tissue culture seedling reaches 100 percent.
Description
Technical field
The present invention relates to plant tissue culture technique, particularly to method and the culture medium thereof of a kind of Vaccinium uliginosum tissue-culturing rapid propagation.
Background technology
Vaccinium uliginosum (Vaccinium uliginosum), having another name called all Fructus Kaki, pasture fruit etc., be edible coloured berry of a kind of Ericaceae (Ericaceae) genus vaccinium (Vaccinium), its fruit is dark blue, delicate mouthfeel, fruital is pleasant.Research shows, Vaccinium uliginosum has the effects such as antioxidation, defying age, anticancer, prevention cardiovascular disease, protection vision, has splendid health-care effect.In China, Vaccinium uliginosum integrated distribution is in the big Xiaoxinanlin Mountains and Changbaishan area, and wherein Daxinganling District is the natural distributed district of its maximum.Investigation display, the natural distributed area of Daxinganling District Vaccinium uliginosum has more than 150,000 hectares, year reserves about 20,000 tons.But, under the driving of interests, the wild Vaccinium uliginosum of China is developed by damage type.The mankind's often destructive Vaccinium uliginosum fruit of gathering in the way of folding branch strip leaf, causes irreversible damage to its natural habitat.It is reported, the wild Vaccinium uliginosum of China occurs in that the phenomenon that Resource Degradation, population area reduce the most prematurely, and Vaccinium uliginosum can list endangered plants monoid in.Therefore, it is necessary to take measures, set up Vaccinium uliginosum protection of resources system.Wherein, exploitativeness is strong, the protective measure of instant effect is set up Vaccinium uliginosum resource exactly and returned the project of planting, i.e. artificial culture's Vaccinium uliginosum nursery stock, and the corresponding original district destroyed by its Hui Zhi to resource; to ensure the sustainability of development of resources, conserve forests ecologic structure.
At present, the foundation of existing research report Vaccinium uliginosum regenerating system, existing system can realize the regeneration of Vaccinium uliginosum.But, owing to Vaccinium uliginosum tissue cultured seedling stem stalk is the most very thin, and more difficulty of taking root in its bottle, take root time-consuming long (usually 40 90 days), and root is thinner and more delicate.Meanwhile, Vaccinium uliginosum tissue cultured seedling is transplanted correlational study and is more short of, and survival rate is relatively low.This present situation is invisible adds Vaccinium uliginosum seedling cost, significantly limit the process of Vaccinium uliginosum industrial seedling rearing, constrains Vaccinium uliginosum protection of resources effect.
Summary of the invention
In order to make up the deficiency in above field, the invention provides method and the culture medium thereof of a kind of Vaccinium uliginosum tissue-culturing rapid propagation.
Culture medium for obtaining Vaccinium uliginosum regeneration plant provided by the present invention, 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, chlorocholine chloride, sucrose and distilled water；
Above composition concentration in described culture medium is respectively as follows:
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, chlorocholine chloride 0mg/L-10mg/L and sucrose 20g/L.
The described culture medium for obtaining Vaccinium uliginosum 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, chlorocholine chloride, sucrose and distilled water；
Above composition concentration in the described culture medium for obtaining Vaccinium uliginosum regeneration plant is respectively as follows:
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, chlorocholine chloride 5mg/L and sucrose 20g/L.
Present invention also offers a kind of solid medium for obtaining Vaccinium uliginosum regeneration plant, be by coagulator and the described solid medium being made into for the culture medium obtaining Vaccinium uliginosum regeneration plant.
Described coagulator concentration in described solid medium is 9g/L；
Described coagulator is agar.
The pH value of described culture medium is 5.4.
Present invention also offers a kind of method obtaining Vaccinium uliginosum regeneration plant, comprise the steps:
It is used for obtaining on the solid medium of Vaccinium uliginosum regeneration plant described in Vaccinium uliginosum regrowth is inoculated into and carries out root induction, i.e. obtain Vaccinium uliginosum regeneration plant.
Described method also includes described Vaccinium uliginosum Transplantation of Regenerated Plantlets to equipped with in cultivation matrix Seedling basin, by the step of Seedling bag auxiliary transplanting.
Described Seedling bag be top with self-sealed article, transparent, the rigid plastics pocket of lower openings, its underpart opening is consistent with Seedling basin caliber size；
The auxiliary transplanting of described Seedling bag is to entangle Seedling basin with Seedling bag, seals the self-sealed article on Seedling bag top, and ties Seedling bag bottom with rubber band, it is ensured that the sealing of this planting environment；
Described cultivation matrix be volume ratio be the turfy soil of 3:1: Vermiculitum.
Described Vaccinium uliginosum regrowth is to be obtained by the method comprised the following steps:
With the induction Vaccinium uliginosum aseptic seedling differentiation of induction culture medium, cultivate and obtain Vaccinium uliginosum regrowth；
Described 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 described culture medium is respectively as follows:
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.
Described 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 described culture medium is respectively as follows:
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.
Generally chlorocholine chloride can impose outward restriction crop overgrowing, has the effect promoting to bear fruit.For the thin and delicate phenomenon of Vaccinium uliginosum nursery stock, present invention firstly provides and chlorocholine chloride has been added culture medium, coordinated phytohormone, the method promoting Vaccinium uliginosum tissue cultured seedling rejuvenation, the method can strengthen its anti-adversity ability by strengthening tissue cultured seedling degree of lignification, to improve the survival rate after nursery stock transplanting.
Further, since Vaccinium uliginosum tissue cultured seedling transplanting initial stage, its root system and branch and leaf are still in recovery, laundering period, its phylactic power defensive power to external world is more weak, and particularly water holding capacity is poor.Therefore, it is necessary to the less stable environment that provides that air humidity is higher, air flows is to ensure Vaccinium uliginosum tissue cultured seedling transplant survival.Between above-mentioned background, the present invention proposes a kind of low cost, the making of reusable and easy to operate Seedling bag and uses (Fig. 3).This Seedling bag can ensure that Vaccinium uliginosum tissue cultured seedling transplants the air-flow of environment and stablizing of humidity, takes root, method in strong sprout in coordinating the Vaccinium uliginosum bottle that the present invention proposes, and Vaccinium uliginosum tissue cultured seedling transplanting survival rate can be made to reach 100%.
Accompanying drawing explanation
Fig. 1 is the different culture media induction situation to Vaccinium uliginosum tissue cultured seedling.
Fig. 2 is the different culture media root induction situation to Vaccinium uliginosum tissue cultured seedling；Wherein A is comparison；B is for adding IBA 0.1mg/L；C is for adding IBA 0.5mg/L；D is for adding IBA 0.5mg/L+ chlorocholine chloride 5mg/L.
Fig. 3 is the Seedling bag that proposes of the present invention and nursery effect thereof；Wherein, A is for cultivating with Seedling bag button basin after transplanting；B is the growth conditions after cultivating 2 months.
Fig. 4 is the growing state after Vaccinium uliginosum tissue cultured seedling is transplanted 2 months；Wherein, A is i.e. Raising strong seedli of taking root in not carrying out bottle；B be take root in bottle after Raising strong seedli；C be take root in bottle after transplant by Seedling bag auxiliary.
Detailed description of the invention
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 (naphthalene acetic acid, 1-Naphthaleneacetic acid), 6BA (6-Benzylaminopurine), chlorocholine chloride, agar powder, sucrose be purchased from Beijing company limited of Kechuang Chendu XinDa, remaining reagent indicated the most especially is all purchased from Beijing chemical company.
Experiment material: Vaccinium uliginosum aseptic seedling.Wild Vaccinium uliginosum picks up from Daxinganling District, is outer implant with the tender stem of giving birth to then of its strong branch, by tissue culture technique, induction axillalry bud regeneration, it is thus achieved that aseptic tissue cultured seedling is used for subsequent experimental.This aseptic seedling can obtain with genetic breeding research group from Institute of Botany, Chinese Academy of Sciences's flowers physiology.The non-patent literature recording Vaccinium uliginosum (Daxing'an Mountainrange district wild species) is: turn round and look at relation by marriage, He Shanan, what rainbow, Wang Chuanyong, the blue berry of Wu Wenlong (2001) and cranberry (the 1st edition). Beijing: Chinese agriculture publishing house .pp.25-33,142-187.
The efficient tissue-culturing rapid propagation of embodiment 1, Vaccinium uliginosum and the method for transplanting
One, preparation Vaccinium uliginosum inductive differentiation medium and root media
1, preparation improvement WPM0Basal medium:
Measure 50mL improvement WPM a great number of elements mother solution, 5mL improvement WPM trace element mother solution, 5mL improvement WPM Organic substance mother solution, 5mL improves WPM mother liquid of iron salt, 20mg sucrose, mix, and with sulphuric acid or phosphorus acid for adjusting pH to 5.4, and constant volume final volume is to 1000ml；Add 9g agar, 121 DEG C of high temperature sterilize 20min, cool to about 65 DEG C, standby.
The improvement WPM mother solution formula that the present embodiment is prepared is as shown in table 1:
The improvement WPM mother solution formula that table 1 the present embodiment is prepared
Remarks: all of reagent is all placed in 4 DEG C of Refrigerator stores after preparing.
2, the hormone of Vaccinium uliginosum induction and root induction it is formulated for
Weigh 10mg ZT (zeatin respectively, Zeatin), IBA (indolebutyric acid, Beta-indolebutyric acid), 2,4D (2,4-Dichlorophenoxyacetic acid), TDZ (Thidiazuron, Thidiazuron), NAA (naphthalene acetic acid, 1-Naphthaleneacetic acid), 6BA (6-Benzylaminopurine) powder, and be settled to 10ml with straight alcohol respectively；Weigh 10mg chlorocholine chloride powder, be settled to 10ml with distilled water；By above-mentioned hormone in super-clean bench, with 0.22 μm filter filtration sterilization.Hormone after sterilizing is placed in 4 DEG C of storages, standby.Hereafter all operations is all carried out in super-clean bench, and used tool is 121 DEG C, the article after sterilizing 20min.
3, preparation is for the culture medium of Vaccinium uliginosum induction
(1) by sterilized basal medium improvement WPM in super-clean bench0After mixing, subpackage is in tissue culture bottle, every bottle of about 50ml；
(2) according to table 2, in the culture medium that subpackage is good, add corresponding hormone, after mixing, stand, use after it solidifies；Every kind of culture medium prepares at least 3 bottles；
The induction culture medium prescription that table 2 the present embodiment is prepared
Improvement 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 at improvement WPM0In basal medium, concentration is respectively as follows:
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。
4, preparation is for the culture medium of Vaccinium uliginosum root induction
(1) by sterilized basal medium improvement WPM in super-clean bench0After mixing, subpackage is in tissue culture bottle, every bottle of about 50ml；
(2) according to table 3, in the culture medium that subpackage is good, add corresponding hormone, after mixing, stand, use after it solidifies；Every kind of culture medium prepares at least 3 bottles；
The root induction culture medium prescription that table 3 the present embodiment is prepared
Improvement 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 at improvement WPM0In basal medium, concentration is respectively as follows:
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。
Two, the induction of Vaccinium uliginosum
(1) after the cooling of induction culture medium, solidification, cut Vaccinium uliginosum aseptic seedling, be respectively connected to the Vaccinium uliginosum induction culture medium of table 2；
(2) tissue cultured seedling is positioned over 20 (± 5) DEG C, 1200lx, cultivates 30 40d under the conditions of periodicity of illumination 16h/d, induce it to break up, obtain Vaccinium uliginosum regeneration tissue cultured seedling.
Three, the root culture of Vaccinium uliginosum tissue cultured seedling
(1) with sharp shears, the Vaccinium uliginosum of the most about 3-4cm being regenerated tissue cultured seedling to cut, be inserted vertically in Vaccinium uliginosum root induction culture medium, every bottle graft enters 30 strains；
(2) postvaccinal tissue culture bottle is placed in 20 (± 5) DEG C, 1200lx, under the conditions of periodicity of illumination 16h/d, cultivates statistics analytical data after 30 40d.
Four, Vaccinium uliginosum is taken root the transplanting of Seedling
(1) take out the Vaccinium uliginosum tissue cultured seedling taken root, clean the culture medium (noticing that this cleaning step needs light and slow operation, be completely cured with disposable) of its root with flowing water；
(2) transplantation of seedlings to cultivation matrix of being taken root by clean Vaccinium uliginosum is turfy soil: (watered by cultivation matrix permeable in advance) in the Seedling basin of Vermiculitum=3:1, carrying out corresponding overlay film by table 4 or set Seedling bag processes, every kind processes including at least 100 young plants；Nursery stock after field planting is placed in 20 (± 5) DEG C, 1200lx, under the conditions of periodicity of illumination 16h/d, cultivates statistics analytical data after 30 40d.
Method is managed and protected in the transplanting that table 4 the present embodiment is carried out
Five, result
(1) thin and little due to Vaccinium uliginosum blade, it is easy to brownization in operation, dries up, and therefore compared to blade, the tender stem of Vaccinium uliginosum is more suitable for as outer implant, and evoking adventive bud regenerates.Experimental result (Fig. 1, table 5) shows have important function for the induction of Vaccinium uliginosum, TDZ and zeatin (ZT), and 2,4D is invalid.NAA or IBA adding low concentration in the culture medium containing 2.0mg/L TDZ can effectively facilitate Vaccinium uliginosum differentiation.The while of present invention demonstrates that, culture medium DSF1 added with TDZ and IBA can promote that culture efficiently breaks up, wherein incision and any tip segment contacted with culture medium of the outer implant of Vaccinium uliginosum all has the callus spreading all over red bud point to generate, and has also germinated a large amount of regeneration bud at the axillalry bud of outer implant.Culture medium DSF2 added with TDZ and NAA also can induce a large amount of callus to generate simultaneously, but this wound healing the most only produces in incision, and differentiation and regeneration effect is not as good as DSF1.But the regrowth in DSF1 and DSF2 culture medium all exists poor growth, the problem that elongation is difficult, need to change the subculture medium that nursery stock can be promoted to extend in time.And the plants such as Fructus Lycii being had to the 2 of preferable induction effect, 4D is not then suitable for Vaccinium uliginosum, and the outer implant being connected in this culture medium cannot break up generation regeneration bud, cannot germinate axillalry bud yet, majority death the most aging, withered.
Table 5 Vaccinium uliginosum induction result
ZT has efficient differentiation-inducing action, is commonly used for the induction of the plant of difficult differentiation.The study show that the differentiation rate of Vaccinium uliginosum increases the most therewith along with ZT concentration changes from 0.5 2.5mg/L.The zeatin (≤0.5mg/L) of low concentration can promote outer implant axillary bud sprouting, and its elongation speed well, and outer implant and culture medium contact position can form a small amount of callus；The zeatin (1 2.5mg/L) of higher concentration breaks up with can stimulating outer planting high levels, and in the culture medium of the final concentration of 2.5mg/L of ZT, the Extending culture time, the differentiation rate of Vaccinium uliginosum can surpass 100.But, along with the raising of level of differentiation, differentiation Seedling occurs that vitrified probability is also increasing, and the tissue cultured seedling stem stalk that the differentiation of high concentration zeatin obtains is the most very thin, is difficult to transplant survival.Therefore, the zeatin of 1.0mg/L is the optium concentration of induction Vaccinium uliginosum differentiation, and the IBA (0.1 0.5mg/L) being added to low concentration has act on certain strong sprout, the very thin degree of nursery stock can be reduced, but the IBA of high concentration can suppress the differentiation of outer implant and elongation.It addition, compared with the culture medium added with TDZ, Vaccinium uliginosum can be induced efficiently to break up a large amount of regeneration buds of generation added with the culture medium of ZT, and this regeneration bud can rapid elongation simultaneously.Therefore the culture medium added with ZT can reduce the complicated degree of culture medium preparation work simultaneously in order to successive transfer culture, shorten the Vaccinium uliginosum tissue culture cycle.To sum up, the improvement WPM of ZT 1.0mg/L and IBA 0.1mg/L it is added with0Culture medium (i.e. culture medium DSF9) is Vaccinium uliginosum optimal induction culture medium.
(2) IBA (0.1 0.5mg/L) of low concentration can effectively facilitate in Vaccinium uliginosum tissue cultured seedling carries out bottle and take root, it is taken root and has obvious inhibiting effect by the IBA (1.0 2.0mg/L) of high concentration, and IBA concentration is the highest, inhibitory action the most obvious (Fig. 2, table 6).Additionally, due to Vaccinium uliginosum tissue cultured seedling is the most very thin, degeneration-resistant level is poor, and for increasing survival rate when it is transplanted, this research with the addition of the chlorocholine chloride of doses in its root media, assists strong sprout, reduces nursery stock overgrowing, promotes that its stem stalk is sturdy.Result shows when IBA concentration level is relatively low (0.1mg/L), and interpolation chlorocholine chloride can significantly inhibit and take root simultaneously, makes rooting rate reduce more than 50%；When IBA concentration level is higher (1.0mg/L), the inhibition simultaneously adding chlorocholine chloride then will not be the most obvious；And when IBA level moderate (0.5mg/L), the chlorocholine chloride adding low dosage with hestening rooting, and can effectively facilitate the increase of tissue cultured seedling degree of lignification.Therefore this research filters out improvement WPM0It is its optimal root induction culture medium (SG8) that basal medium adds IBA 0.5mg/L+ chlorocholine chloride 5mg/L, and rooting rate is 81.47 ± 3.48%.
Table 6 Vaccinium uliginosum tissue cultured seedling root induction result
(3) this research to take root in not carrying out bottle, the Vaccinium uliginosum tissue cultured seedling in the strong sprout transplanting survival rate that directly carries out transplanting as comparison, Raising strong seedli and the transplanting effect with Seedling bag (Fig. 3) auxiliary transplanting after taking root in contrast bottle respectively.Survival rate is added up after transplanting 30 days, result (Fig. 4) shows, take root in not carrying out bottle, its transplanting survival rate of Vaccinium uliginosum tissue cultured seedling in strong sprout is up to 69.37 ± 4.95%, after taking root in Ping, the tissue cultured seedling survival rate of Raising strong seedli is 73.79 ± 3.00%, and with the plant percent of Seedling bag auxiliary transplanting up to 100%.
It should be noted that, seedling growth situation is again observed after transplanting 60 days, its survival rate of tissue cultured seedling taken root in finding not carry out bottle drops to 54.89 ± 4.01%, nearly 15% is have dropped than the data of 1 each moon after transplanting, and plant height change is seldom, almost do not increase, but the tissue cultured seedling after taking root in bottle shows as stable growth.Simultaneously, originally research and propose for assisting the Seedling bag of transplanting can maintain the stability of tissue cultured seedling growth environment to a great extent, including air humidity, temperature and air flow property, this gradually adapts to extraneous growing environment after being conducive to tissue cultured seedling bottle outlet, provide preferable spatial transition, drastically increase plant percent, and plant strain growth speed.In this research, transplant more than 150 strain Vaccinium uliginosum tissue cultured seedlinies with described Seedling bag auxiliary method for transplanting, the most survived, and come along fine.
Result shows, the Vaccinium uliginosum division culture medium that the present invention proposes can induce tissue cultured seedling efficiently to break up, and while in the Vaccinium uliginosum tissue cultured seedling bottle adding chlorocholine chloride and IBA in the medium of present invention proposition, rooting method is taken root in promoting Vaccinium uliginosum bottle (rooting rate is up to more than 80%), strong sprout can be realized, the degree of lignification of Vaccinium uliginosum tissue cultured seedling is greatly improved, anti-adversity ability when Vaccinium uliginosum tissue cultured seedling is transplanted can be effectively increased, and the method is simple to operate, application is strong.Meanwhile, the inventive method proposes the method that Seedling bag assisted group seedlings cultivating is transplanted, and the method is capable of the transplanting survival rate of Vaccinium uliginosum tissue cultured seedling 100%, if for industrial seedling rearing, enterprise planting percent can be greatly improved, reduce entreprise cost, the using value having had.Meanwhile, the design of this Seedling bag is simple, easy to operate, and can reuse.Additionally, this Seedling bag is applicable to variously-shaped Seedling basin utensil, it is also possible to for the cultivation of other rare materials, there is preferable promotional value.Generally, the transplantation of seedlings cycle that goes out of Vaccinium uliginosum tissue cultured seedling is foreshortened to 30 40d (counting from root culture) from more than 60d by the present invention, and complete process in strong sprout while induction tissue cultured seedling is taken root, with the use of Seedling bag, Vaccinium uliginosum tissue cultured seedling transplanting survival rate is improved to 100%, so, this method for Vaccinium uliginosum industrial seedling rearing and other tissue cultured seedling wood is thin and delicate, difficulty of taking root and to transplant the difficulty cultivation of plant such as live significant.
Claims (10)
1., for obtaining a culture medium for Vaccinium uliginosum regeneration plant, consist 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, chlorocholine chloride, sucrose and distilled water；
Above composition concentration in described culture medium is respectively as follows:
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, chlorocholine chloride 0mg/L-10mg/L and sucrose 20g/L.
Culture medium for obtaining Vaccinium uliginosum regeneration plant the most according to claim 1, it is characterised in that: described training
Foster base 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, chlorocholine chloride, sucrose and distilled water；
Above composition concentration in described culture medium is respectively as follows:
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, chlorocholine chloride 5mg/L and sucrose 20g/L.
3. for obtaining a solid medium for Vaccinium uliginosum regeneration plant, described in coagulator and claim 1 or 2
The solid medium that culture medium is made into.
Solid medium the most according to claim 3, it is characterised in that:
Described coagulator concentration in described solid medium is 9g/L；
Described coagulator is agar.
5. according to described culture medium arbitrary in claim 1-4, it is characterised in that:
The pH value of described culture medium is 5.4.
6. the method obtaining Vaccinium uliginosum regeneration plant, comprises the steps:
Vaccinium uliginosum regrowth is inoculated in claim 3-5 and carries out root induction in arbitrary described culture medium, i.e. obtain sincere
This Pericarpium Citri tangerinae regeneration plant.
Method the most according to claim 6, it is characterised in that:
Described method also includes described Vaccinium uliginosum Transplantation of Regenerated Plantlets to equipped with in cultivation matrix Seedling basin, moves by Seedling bag auxiliary
The step planted.
Method the most according to claim 7, it is characterised in that:
Described Seedling bag is that top is with self-sealed article, transparent, the rigid plastics pocket of lower openings, its underpart opening and Miao Penkou
Footpath is in the same size；
The auxiliary transplanting of described Seedling bag is to entangle Seedling basin with Seedling bag, seals the self-sealed article on Seedling bag top, and pricks with rubber band
Live Seedling bag bottom, it is ensured that the sealing of this planting environment；
Described cultivation matrix be volume ratio be the turfy soil of 3:1: Vermiculitum.
Method the most according to claim 6, it is characterised in that:
Described Vaccinium uliginosum regrowth is to be obtained by the method comprised the following steps:
With the induction Vaccinium uliginosum aseptic seedling differentiation of induction culture medium, cultivate and obtain Vaccinium uliginosum regrowth；
Described 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 described culture medium is respectively as follows:
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.
Method the most according to claim 9, it is characterised in that:
Described 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 described culture medium is respectively as follows:
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.
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