CN107135950A - A kind of breeding method of quick acquisition black fruit fructus lycii regrowth - Google Patents
A kind of breeding method of quick acquisition black fruit fructus lycii regrowth Download PDFInfo
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- CN107135950A CN107135950A CN201710463521.4A CN201710463521A CN107135950A CN 107135950 A CN107135950 A CN 107135950A CN 201710463521 A CN201710463521 A CN 201710463521A CN 107135950 A CN107135950 A CN 107135950A
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- fructus lycii
- black fruit
- fruit fructus
- culture
- regrowth
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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
- A01H4/008—Methods for regeneration to complete plants
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01C—PLANTING; SOWING; FERTILISING
- A01C1/00—Apparatus, or methods of use thereof, for testing or treating seed, roots, or the like, prior to sowing or planting
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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
- A01H4/001—Culture apparatus for tissue culture
Abstract
The invention belongs to forest tree biotechnology field, a kind of breeding method of quick acquisition black fruit fructus lycii regrowth is disclosed, following steps are specifically included：The sterilizing of seed, the acquisition of sterile seedling, the induction of callus, the acquisition of adventitious bud and the induction of root.It is of the invention that callus is directly obtained by the sterile seedling of black fruit fructus lycii, black fruit fructus lycii regrowth is cultivated by method for tissue culture, compared with the blade or stem section evoked callus that tradition passes through seedling, the present invention can greatly shorten the time of tissue culture, considerably reduce the cultivation cost of black fruit fructus lycii, cultivation efficiency is also effectively increased simultaneously, and the transplanting survival rate of regrowth is up to 98%.The black fruit fructus lycii that the present invention is obtained has that genetic stability is good, cost is low, growing-seedling period is short, breeding coefficient is high, it is easy to operate the characteristics of.
Description
Technical field
The invention belongs to forest tree biotechnology field, it is related to a kind of plant cultivating method, and in particular to a kind of quick acquisition
The breeding method of black fruit fructus lycii regrowth.
Background technology
Black fruit fructus lycii (Lycium Ruthenicum Murr), is the perennial many quils of Solanaceae (Solanceae) Lycium
Shrub plant, is one of important drought resisting, salt resistance alkali desert vanguard tree seed, it is in China Tibet, Helan Mountain in Ningxia, Qing Haidong
All there is fragmentary distribution in the suitable rugged environment of the condition such as portion, North Shaanxi, In The North of Xinjiang, interior ridge, riverbed sandy beach.
Wild lycium ruthenicum is sweet, mild-natured, rich in protein, fat, carbohydrate, free amino acid, organic acid, mineral matter, micro-
The various nutrition such as secondary element, alkaloid, vitamin C, B1, B2, calcium, magnesium, copper, zinc, manganese, iron, lead, nickel, cadmium, cobalt, chromium, potassium, sodium
Composition.Detected through science, the calcium, iron, niacin content in black fruit fructus lycii be respectively haw matrimony vine 2.3,4.6,16.7 times, especially
It is that OPC (OPC) content is even more than blueberry (black fruit fructus lycii 3690mg/100g containing OPC；Blueberry containing OPC 330~
3380mg/100g), it is to find OPC content highest wild fruits so far, is also maximally effective natural anti-oxidation
Agent, its effect is 20 times of VC, 50 times of VE, the tonic effect with superpower strengthen immunity, anti-aging.Black fruit fructus lycii
In the nutrient composition content such as vitamin, mineral matter it is also very abundant, especially containing with remove free radical, anti-oxidation function it is natural
Anthocyanin element, medicinal, health value is significantly larger than common red matrimony vine, is described as " soft gold ".
Black fruit fructus lycii integrates economic value, the ecological value, medical value and health value, and its price occupies height not always
Under.In recent years, the harvesting of agriculture and animal husbandry madness allows some wild black fruit fructus lyciis no longer to bear fruit.How black fruit Chinese holly is adequately and reasonably developed
Qi resource, is the important topic for needing concern badly.Current black fruit fructus lycii nursery stock markets are still based on seedling, but seedling price is higher,
And be difficult the merit for keeping parent completely.
The tissue cultures of plant are theoretical asexual in one grown up in recent decades according to totipotency of plant cell
The new technology of breeding.Because Plant Tissue Breeding have occupy little space, not by area and season limit, cultivation cycle it is short, can be short
Time amount reproduction, in the absence of variation, the advantages such as maternal all hereditary features and small investment, economic benefit height can be kept,
So that the technology is once appearance, it is just rapid to be used widely in the production of flowers, crops and achievements in forest-tree seedling.Therefore, by
The tissue culture technique of plant, sets up the breeding method of black fruit fructus lycii regrowth, with large-scale production black fruit fructus lycii seedling, so that
Meet the harvesting high-quality high-yield and industrialization needs of black fruit fructus lycii.
For example：Chinese patent literature CN105850733A discloses a kind of black fruit fructus lycii regeneration seeding cultivating method, and its is direct
Adventitious bud is obtained by the blade of black fruit fructus lycii or Multiple Buds are obtained by the stem section of black fruit fructus lycii, because this method needs head
The blade or adventitious bud of black fruit fructus lycii are first obtained, therefore tissue culture is of long duration, and bud formation rate is not high.And for example Chinese patent literature
CN105815221A discloses a kind of black fruit fructus lycii method for in-vitro rapid propagation using young seedling as explant donor, and it passes through
Acquisition, explant (blade or stem section of seedling) inoculation, shoot proliferation and acclimatization and transplantses of sterile seedling etc. are completed, and this method is real
Matter is also to obtain Multiple Buds by the blade or stem section of black fruit fructus lycii, and it is 10 to need to wait seedling to grow to the number of blade in the process
It can carry out the inoculation of explant when~20, and when being that bud formation rate when explant is inoculated with one month is using kind of seedling leaf
87.5%.As can be seen here, the existing black fruit fructus lycii regeneration seeding cultivating method generally existing tissue culture time is long, and bud formation rate is not high
Defect.
The content of the invention
Therefore, the technical problem to be solved in the present invention is to overcome the cultivation for obtaining black fruit fructus lycii regrowth in the prior art
Time length and the not high defect of the bud formation rate obtained by explant, so as to provide a kind of quick acquisition black fruit fructus lycii regrowth
Breeding method.
In order to solve the above technical problems, the technical solution adopted by the present invention is specific as follows：
A kind of breeding method of quick acquisition black fruit fructus lycii regrowth, this method is the sterile seedling directly by black fruit fructus lycii
Callus is obtained, then black fruit fructus lycii regrowth is obtained through tissue cultures cultivation, it comprises the following steps：
(1) sterilization of seed
Choose black fruit fructus lycii seed, successively sterilized dose sterilization and aseptic water washing after it is standby；
(2) acquisition of sterile seedling
The seed of step (1) is placed in MS-0 culture mediums, sterile seedling is obtained after the seed is sprouted 8~12 days；
The MS-0 culture mediums are obtained, the MS- by adding 30g sucrose and 8.0g agar into every liter of MS culture medium
The pH value of 0 culture medium is 5.0~5.5；
(3) induction of callus
By sterile kind of transplantation of seedlings of step (2) into MS-1 culture mediums with induced synthesis callus, condition of culture is：Temperature
23~27 DEG C of degree, illumination 12h/d, Medium's PH Value are 5.0~5.5, are cultivated 35~45 days；
The MS-1 culture mediums are by adding 0.5~1.0mgNAA, 1.5~2.5mg 6- into every liter of MS culture medium
BA, 30g sucrose and 8.0g agar and obtain；
(4) acquisition of adventitious bud
Break up indefinite during the callus of step (3) is placed in into MS-2 culture mediums after being stripped down on explant to induce
Bud, condition of culture is：23~27 DEG C of temperature, illumination 14h/d, Medium's PH Value are 5.0~5.5, the generation of Multiplying culture 3~5；
The MS-2 culture mediums are by adding 0.5~1.0mgNAA, 0.5~1.4mg 6- into every liter of MS culture medium
BA, 30g sucrose and 6.0g agar and obtain；
(5) induction of root
The adventitious bud of step (4) is transplanted in the MS-0 culture mediums with root induction, black fruit fructus lycii regrowth is produced；
Condition of culture is：23~27 DEG C of temperature, illumination 14h/d, incubation time 17~22 days.
Preferably, the sterilizing part in step (1) is first with 70v% alcohol-pickled 3~7min, then to use 0.1wt%
Mercuric chloride solution sterilize 10~20min.
It is further preferred that the time of the mercuric chloride solution sterilization is 15min.
Preferably, the compound method of the MS culture mediums includes, by the micro- mother liquors of MS, MS inositols mother liquor, MS iron
Salt mother liquor dilutes after 100 times respectively to be mixed with diluting 40 times of MS a great number of elements mother liquors；
The ratio between the micro- mother liquors of the MS, MS inositols mother liquor, MS mother liquid of iron salt, volume of MS a great number of elements mother liquors are 5:
5:5:2；
The composition of the MS a great number of elements mother liquor is to contain KNO in every liter of sterilized water3 76.0g、NH4NO3 66.0g、
MgSO4·7H2O 14.8g、CaCl2·2H2O 17.6g, anhydrous CaCl213.28g；
The composition of the micro- mother liquors of the MS is to contain MnSO in every liter of sterilized water4·H2O 2.23g、ZnSO4·7H2O
0.86g、H3BO3 0.62g、KI 0.083g、Na2MoO4·2H2O 0.025g、CuSO4·5H2O 0.0025g、CoCl2·6H2O
0.0025g；
The composition of the MS inositols mother liquor is, 10.0g containing inositol, nicotinic acid 0.1g, Vit B in every liter of sterilized water1 1.0g、
Vit B60.1g；
The composition of the MS mother liquid of iron salt is, FeSO4·7H2O 2.78g、Na2EDTA 3.73g。
Preferably, the MS-0 culture mediums, MS-1 culture mediums, the pH value of MS-2 culture mediums are 5.2.
Preferably, 1.0mg containing NAA, 6-BA 2.0mg in every liter of MS-1 culture medium.
Preferably, 1.0mg containing NAA, 6-BA 1.0mg in every liter of MS-2 culture medium.
Preferably, the breeding method of described a kind of quick acquisition black fruit fructus lycii regrowth, in addition to, by step (5)
Obtained rooted seedling is transferred to the hardening step of at least 2 weeks in greenhouse.
V% in the present invention represents volumn concentration, and wt% represents weight/mass percentage composition.
Technical scheme, has the following advantages that：
(1) breeding method for the quick acquisition black fruit fructus lycii regrowth that the present invention is provided, directly by the sterile of black fruit fructus lycii
Seedling obtains callus, then cultivates black fruit fructus lycii regrowth through tissue cultures, and method of the invention reduces the life of aseptic seedling
Long link, compared with the blade or stem section evoked callus that tradition passes through seedling, the present invention can greatly shorten the time of tissue culture,
The cultivation cost of black fruit fructus lycii is considerably reduced, while also effectively increasing cultivation efficiency, the transplanting survival rate of regrowth is high
Up to 98%.
(2) breeding method for the quick acquisition black fruit fructus lycii regrowth that the present invention is provided, by into every liter of MS culture medium
Add 0.5~1.0mgNAA and 1.5~2.5mg 6-BA, it is easier to obtain the callus of differentiation, and callus production rate is up to
To 94%.
(3) breeding method for the quick acquisition black fruit fructus lycii regrowth that the present invention is provided, by into every liter of MS culture medium
Add 0.5~1.0mgNAA and 0.5~1.4mg 6-BA, be conducive to the differentiation of adventitious bud, can fast germination, and bud ratio reaches
More than 92%.
(4) breeding method of quick acquisition black fruit fructus lycii regrowth that the present invention is provided, in root induction without appointing
What hormone can also ensure that rooting rate is up to 97%, and the speed of growth of root is very fast, and every plant of seedling at least grows 3 roots, and root system
Sturdy, root hair is dense.
(5) breeding method for the quick acquisition black fruit fructus lycii regrowth that the present invention is provided, the sterilization of seed uses 0.1wt%
Mercuric chloride solution sterilization 15min, very well, and pollution rate and the death rate are all very low, respectively 6.5% He for Disinfection Effect
8.5%.
(6) black fruit fructus lycii that the breeding method for the quick acquisition black fruit fructus lycii regrowth that the present invention is provided is cultivated, which has, loses
Pass stability it is good, with low cost, growing-seedling period is short, breeding coefficient is high, it is easy to operate the characteristics of.
Brief description of the drawings
, below will be to specific in order to illustrate more clearly of the specific embodiment of the invention or technical scheme of the prior art
The accompanying drawing used required in embodiment or description of the prior art is briefly described, it should be apparent that, in describing below
Accompanying drawing is some embodiments of the present invention, for those of ordinary skill in the art, before creative work is not paid
Put, other accompanying drawings can also be obtained according to these accompanying drawings.
Fig. 1 be it is according to embodiments of the present invention 1 provide quick acquisition black fruit fructus lycii regrowth breeding method obtain it is black
The growth conditions schematic diagram of fruit matrimony vine plant regeneration system, wherein, A:Aseptic seedling is planted 1 week or so, callus growth situation；
B:Aseptic seedling is planted 2 weeks or so, callus growth situation；C:Aseptic seedling is planted 3 weeks or so, callus growth situation；D:
After callus is peeled off, callus growth about 20 days；E:After callus is peeled off, callus growth about 30 days；F:After callus is peeled off, more
Injured tissue grows about 40 days；G:Occur multiple differentiation budlets on callus；H:The differentiation seedling of continued growth on callus；
I:Start the lycium ruthenicum seedling of culture of rootage.
Embodiment
Technical scheme will be clearly and completely described below, it is clear that described embodiment is this hair
Bright a part of embodiment, rather than whole embodiments.Based on the embodiment in the present invention, those of ordinary skill in the art are not having
There is the every other embodiment made and obtained under the premise of creative work, belong to the scope of protection of the invention.In addition, below
Just can be mutual as long as the technical characteristic involved by described different embodiments of the present invention does not constitute conflict each other
With reference to.
Experimental example 1：MS culture mediums are configured
S1：MS culture medium mother liquors are configured
Table of comparisons 1-4, weighs each component respectively.
Table 1：MS a great number of elements mother liquor is formulated
After weighing is finished, respectively with mixing, last constant volume to 1L after a small amount of distilled water thoroughly dissolving.
Table 2：MS trace element mother liquor formulas
After weighing is finished, respectively with mixing, last constant volume to 1L after a small amount of distilled water thoroughly dissolving.
Table 3：MS inositols mother liquor is formulated
After weighing is finished, respectively with mixing, last constant volume to 1L after a small amount of distilled water thoroughly dissolving.
Table 4：MS mother liquid of iron salt is formulated
After weighing is finished, respectively with mixing, last constant volume to 1L after a small amount of distilled water thoroughly dissolving.
S2：MS culture mediums are configured
The micro- mother liquors of MS in S1, MS inositols mother liquor, MS mother liquid of iron salt are diluted after 100 times with diluting 40 respectively
MS a great number of elements mother liquor again is mixed by equal proportion.
Experimental example 2：Hormone NAA and 6-BA configuration
0.04g NAA powder is weighed, is first dissolved with 1ml absolute ethyl alcohols, then constant volume is to obtain 0.2mg/ml's in 200ml
NAA solution, after to be stored in 4 DEG C of refrigerators standby.
0.04g 6-BA powder is weighed, is first dissolved with 1mol/L diluted sodium hydroxide solutions, then constant volume is to obtain in 200ml
0.2mg/ml 6-BA solution, after to be stored in 4 DEG C of refrigerators standby.
Embodiment 1
1. the sterilization of seed
Black fruit fructus lycii seed is chosen, is sterilized with 0.1wt% mercuric chloride solution is used after 70v% alcohol-pickled 5min again
15min, then with aseptic water washing 3 times.
2. the acquisition of sterile seedling
30g sucrose and 8.0g agar are added into every liter of MS culture medium, with 1mol/L NaOH solution adjust pH value to
5.8, afterwards with MS-0 culture mediums are obtained after 121 DEG C of autoclaving 15min, now the pH value of culture medium is 5.2.
The seed of step 1 is placed in MS-0 culture mediums, sterile seedling is obtained after the seed is sprouted 8~12 days.
3. the induction of callus
1.0mgNAA, 2.0mg 6-BA, 30g sucrose and 8.0g agar are added into every liter of MS culture medium, with 1mol/L's
NaOH solution adjusts pH value to 5.8, afterwards with MS-0 culture mediums are obtained after 121 DEG C of autoclaving 15min, now the pH value of culture medium
For 5.2.
By sterile kind of transplantation of seedlings of step 2 into MS-1 culture mediums with induced synthesis callus, after measured, callus generation
Rate is 94%.Refer to figure A~C in Fig. 1.Condition of culture is：23~27 DEG C of temperature, illumination 12h/d, Medium's PH Value are
5.2, cultivate 35~45 days.
4. the acquisition of adventitious bud
1.0mgNAA, 1.0mg 6-BA, 30g sucrose and 6.0g agar are added into every liter of MS culture medium, with 1mol/L's
NaOH solution adjusts pH value to 5.8, afterwards with MS-2 culture mediums are obtained after 121 DEG C of autoclaving 15min, now the pH value of culture medium
For 5.2.
Break up indefinite during the callus of step 3 is placed in into MS-2 culture mediums after being stripped down on explant to induce
Bud, after measured, young shoot production rate are 95%.Figure D~H in Fig. 1 is referred to, condition of culture is：23~27 DEG C of temperature, illumination
14h/d, Medium's PH Value are 5.2, the generation of Multiplying culture 3~5.
5. piece induction
The adventitious bud of step 4 is transplanted in MS-0 culture mediums with root induction, the figure I in Fig. 1 is referred to, after measured,
Rooting rate is 97%.Wherein：Condition of culture is：23~27 DEG C of temperature, illumination 14h/d, incubation time 17~22 days.
6. acclimatization and transplantses
The rooted seedling that step 5 is obtained was transferred in greenhouse hardening after 2 weeks, was carried out using the method for environmental condition gradually transition
Transplant.After measured, transplanting survival rate is 98%.
Embodiment 2
1. the sterilization of seed
Black fruit fructus lycii seed is chosen, is sterilized with 0.1wt% mercuric chloride solution is used after 70v% alcohol-pickled 7min again
10min, then with aseptic water washing 3 times.
2. the acquisition of sterile seedling
30g sucrose and 8.0g agar are added into every liter of MS culture medium, with 1mol/L NaOH solution adjust pH value to
5.6, afterwards with MS-0 culture mediums are obtained after 121 DEG C of autoclaving 15min, now the pH value of culture medium is 5.0.
The seed of step 1 is placed in MS-0 culture mediums, sterile seedling is obtained after the seed is sprouted 8~12 days.
3. the induction of callus
0.5mgNAA, 2.5mg 6-BA, 30g sucrose and 8.0g agar are added into every liter of MS culture medium, with 1mol/L's
NaOH solution adjusts pH value to 5.6, afterwards with MS-0 culture mediums are obtained after 121 DEG C of autoclaving 15min, now the pH value of culture medium
For 5.0.
By sterile kind of transplantation of seedlings of step 2 into MS-1 culture mediums with induced synthesis callus, after measured, callus generation
Rate is 91%.Condition of culture is：23~27 DEG C of temperature, illumination 12h/d, Medium's PH Value are 5.2, are cultivated 35~45 days.
4. the acquisition of adventitious bud
0.5mgNAA, 1.4mg 6-BA, 30g sucrose and 6.0g agar are added into every liter of MS culture medium, with 1mol/L's
NaOH solution adjusts pH value to 5.6, afterwards with MS-2 culture mediums are obtained after 121 DEG C of autoclaving 15min, now the pH value of culture medium
For 5.0.
Break up indefinite during the callus of step 3 is placed in into MS-2 culture mediums after being stripped down on explant to induce
Bud, after measured, young shoot production rate are 94%.Condition of culture is：23~27 DEG C of temperature, illumination 14h/d, Medium's PH Value are 5.0,
The generation of Multiplying culture 3~5.
5. piece induction
The adventitious bud of step 4 is transplanted in MS-0 culture mediums with root induction, after measured, rooting rate is 96%.Wherein：
Condition of culture is：23~27 DEG C of temperature, illumination 14h/d, incubation time 17~22 days.
6. acclimatization and transplantses
After rooted seedling is transferred in greenhouse into hardening 2 weeks, transplanted using the method for environmental condition gradually transition.Through surveying
Fixed, transplanting survival rate is 94%.
Embodiment 3
1. the sterilization of seed
It is again 0.1% with volume fraction to choose after black fruit fructus lycii seed, the alcohol-pickled 3min for being 70% with volume fraction
Mercuric chloride solution sterilization 20min, then with aseptic water washing 3 times.
2. the acquisition of sterile seedling
30g sucrose and 8.0g agar are added into every liter of MS culture medium, with 1mol/L NaOH solution adjust pH value to
6.1, afterwards with MS-0 culture mediums are obtained after 121 DEG C of autoclaving 15min, now the pH value of culture medium is 5.5.
The seed of step 1 is placed in MS-0 culture mediums, sterile seedling is obtained after the seed is sprouted 8~12 days.
3. the induction of callus
0.8mgNAA, 1.5mg 6-BA, 30g sucrose and 8.0g agar are added into every liter of MS culture medium, with 1mol/L's
NaOH solution adjusts pH value to 6.1, afterwards with MS-0 culture mediums are obtained after 121 DEG C of autoclaving 15min, now the pH value of culture medium
For 5.5.
By sterile kind of transplantation of seedlings of step 2 into MS-1 culture mediums with induced synthesis callus, after measured, callus generation
Rate is 90%.Condition of culture is：23~27 DEG C of temperature, illumination 12h/d, Medium's PH Value are 5.5, are cultivated 35~45 days.
4. the acquisition of adventitious bud
0.8mgNAA, 0.5mg 6-BA, 30g sucrose and 6.0g agar are added into every liter of MS culture medium, with 1mol/L's
NaOH solution adjusts pH value to 6.1, afterwards with MS-2 culture mediums are obtained after 121 DEG C of autoclaving 15min, now the pH value of culture medium
For 5.5.
Break up indefinite during the callus of step 3 is placed in into MS-2 culture mediums after being stripped down on explant to induce
Bud, after measured, young shoot production rate are 92%.Condition of culture is：23~27 DEG C of temperature, illumination 14h/d, Medium's PH Value are 5.0,
The generation of Multiplying culture 3~5.
5. piece induction
The adventitious bud of step 4 is transplanted in MS-0 culture mediums with root induction, after measured, rooting rate is 92%.Wherein：
Condition of culture is：23~27 DEG C of temperature, illumination 14h/d, incubation time 17~22 days.
6. acclimatization and transplantses
After rooted seedling is transferred in greenhouse into hardening 2 weeks, transplanted using the method for environmental condition gradually transition.Through surveying
Fixed, transplanting survival rate is 95%.
Comparative example 1
1. the sterilization of seed
Black fruit fructus lycii seed is chosen, is sterilized with 0.1wt% mercuric chloride solution is used after 70v% alcohol-pickled 5min again
15min, then with aseptic water washing 3 times.
2. the acquisition of sterile seedling
30g sucrose and 8.0g agar are added into every liter of MS culture medium, with 1mol/L NaOH solution adjust pH value to
5.8, afterwards with MS-0 culture mediums are obtained after 121 DEG C of autoclaving 15min, now the pH value of culture medium is 5.2.
The seed of step 1 is placed in MS-0 culture mediums, treats that the seed is sprouted to seedling and grows to the number of blade for 10~20
Piece.
3. the induction of callus
1.0mgNAA, 2.0mg 6-BA, 30g sucrose and 8.0g agar are added into every liter of MS culture medium, with 1mol/L's
NaOH solution adjusts pH value to 5.8, afterwards with MS-0 culture mediums are obtained after 121 DEG C of autoclaving 15min, now the pH value of culture medium
For 5.2.
By well-grown spire piece Transverse Shear in step 2 or it is cut to 3~5mm of diameter fritter (monolithic leaf is cut to 2~3
Block) transplant afterwards to planting into MS-1 culture mediums with induced synthesis callus, after measured, callus production rate is 79%.Cultivate bar
Part is：23~27 DEG C of temperature, illumination 12h/d, Medium's PH Value are 5.2, are cultivated 35~45 days.
4. the acquisition of adventitious bud
1.0mgNAA, 1.0mg 6-BA, 30g sucrose and 6.0g agar are added into every liter of MS culture medium, with 1mol/L's
NaOH solution adjusts pH value to 5.8, afterwards with MS-2 culture mediums are obtained after 121 DEG C of autoclaving 15min, now the pH value of culture medium
For 5.2.
Break up indefinite during the callus of step 3 is placed in into MS-2 culture mediums after being stripped down on explant to induce
Bud, after measured, young shoot production rate are 84%.Condition of culture is：23~27 DEG C of temperature, illumination 14h/d, Medium's PH Value are 5.2,
The generation of Multiplying culture 3~5.
5. piece induction
The adventitious bud of step 4 is transplanted in MS-0 culture mediums with root induction, after measured, rooting rate is 87%.Wherein：
Condition of culture is：23~27 DEG C of temperature, illumination 14h/d, incubation time 17~22 days.
6. acclimatization and transplantses
The rooted seedling that step 5 is obtained was transferred in greenhouse hardening after 2 weeks, was carried out using the method for environmental condition gradually transition
Transplant.After measured, transplanting survival rate is 89%.
Obviously, above-described embodiment is only intended to clearly illustrate example, and the not restriction to embodiment.It is right
For those of ordinary skill in the art, can also make on the basis of the above description it is other it is various forms of change or
Change.There is no necessity and possibility to exhaust all the enbodiments.And the obvious change thus extended out or
Among changing still in the protection domain of the invention.
Claims (9)
1. a kind of breeding method of quick acquisition black fruit fructus lycii regrowth, it is characterised in that：Methods described is directly by black fruit Chinese holly
The sterile seedling of Qi obtains callus, then obtains black fruit fructus lycii regrowth through tissue cultures cultivation.
2. the breeding method of quick acquisition black fruit fructus lycii regrowth according to claim 1, it is characterised in that：Methods described
Comprise the following steps：
(1) sterilization of seed
Choose black fruit fructus lycii seed, successively sterilized dose sterilization and aseptic water washing after it is standby；
(2) acquisition of sterile seedling
The seed of step (1) is placed in MS-0 culture mediums, sterile seedling is obtained after the seed is sprouted 8~12 days；
The MS-0 culture mediums are obtained by adding 30g sucrose and 8.0g agar into every liter of MS culture medium, the MS-0 trainings
The pH value for supporting base is 5.0~5.5；
(3) induction of callus
By sterile kind of transplantation of seedlings of step (2) into MS-1 culture mediums with induced synthesis callus, condition of culture is：Temperature 23
~27 DEG C, illumination 12h/d, Medium's PH Value be 5.0~5.5, cultivate 35~45 days；
The MS-1 culture mediums are by adding 0.5~1.0mgNAA, 1.5~2.5mg 6-BA, 30g into every liter of MS culture medium
Sucrose and 8.0g agar and obtain；
(4) acquisition of adventitious bud
To induce differentiation adventitious bud during the callus of step (3) is placed in into MS-2 culture mediums after being stripped down on explant,
Condition of culture is：23~27 DEG C of temperature, illumination 14h/d, Medium's PH Value are 5.0~5.5, the generation of Multiplying culture 3~5；
The MS-2 culture mediums are by adding 0.5~1.0mgNAA, 0.5~1.4mg 6-BA, 30g into every liter of MS culture medium
Sucrose and 6.0g agar and obtain；
(5) induction of root
The adventitious bud of step (4) is transplanted in the MS-0 culture mediums with root induction, black fruit fructus lycii regrowth is produced；
Condition of culture is：23~27 DEG C of temperature, illumination 14h/d, incubation time 17~22 days.
3. the breeding method of quick acquisition black fruit fructus lycii regrowth according to claim 2, it is characterised in that：Step (1)
In sterilizing part be, first with 70v% alcohol-pickled 3~7min, then with 0.1wt% mercuric chloride solution sterilization 10~
20min。
4. the breeding method of the quick acquisition black fruit fructus lycii regrowth according to Claims 2 or 3, it is characterised in that the chlorine
The time for changing mercury solution sterilization is 15min.
5. the breeding method of the quick acquisition black fruit fructus lycii regrowth according to claim any one of 2-4, it is characterised in that：
The compound method of the MS culture mediums includes, and the micro- mother liquors of MS, MS inositols mother liquor, MS mother liquid of iron salt are diluted into 100 respectively
Mixed after times with diluting 40 times of MS a great number of elements mother liquors；
The ratio between the micro- mother liquors of the MS, MS inositols mother liquor, MS mother liquid of iron salt, volume of MS a great number of elements mother liquors are 5:5:5:
2；
The composition of the MS a great number of elements mother liquor is to contain KNO in every liter of sterilized water3 76.0g、NH4NO3 66.0g、MgSO4·
7H2O 14.8g、CaCl2·2H2O 17.6g, anhydrous CaCl213.28g；
The composition of the micro- mother liquors of the MS is to contain MnSO in every liter of sterilized water4·H2O 2.23g、ZnSO4·7H2O
0.86g、H3BO3 0.62g、KI 0.083g、Na2MoO4·2H2O 0.025g、CuSO4·5H2O 0.0025g、CoCl2·6H2O
0.0025g；
The composition of the MS inositols mother liquor is, 10.0g containing inositol, nicotinic acid 0.1g, Vit B in every liter of sterilized water1 1.0g、Vit B6
0.1g；
The composition of the MS mother liquid of iron salt is, FeSO4·7H2O 2.78g、Na2EDTA 3.73g。
6. a kind of breeding method of quick acquisition black fruit fructus lycii regrowth according to claim any one of 2-5, its feature
It is：The MS-0 culture mediums, MS-1 culture mediums, the pH value of MS-2 culture mediums are 5.2.
7. a kind of breeding method of quick acquisition black fruit fructus lycii regrowth according to claim any one of 2-6, its feature
It is：1.0mg containing NAA, 6-BA 2.0mg in every liter of MS-1 culture medium.
8. a kind of breeding method of quick acquisition black fruit fructus lycii regrowth according to claim any one of 2-7, its feature
It is：1.0mg containing NAA, 6-BA 1.0mg in every liter of MS-2 culture medium.
9. a kind of breeding method of quick acquisition black fruit fructus lycii regrowth according to claim any one of 2-8, its feature
It is：The rooted seedling for also including obtaining step (5) is transferred to the hardening step of at least 2 weeks in greenhouse.
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