CN108849499A - A kind of expanding propagation method of the method for preparing Rhizoma Et Radix Notopterygii artificial seed and artificial seed - Google Patents
A kind of expanding propagation method of the method for preparing Rhizoma Et Radix Notopterygii artificial seed and artificial seed Download PDFInfo
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- CN108849499A CN108849499A CN201810522135.2A CN201810522135A CN108849499A CN 108849499 A CN108849499 A CN 108849499A CN 201810522135 A CN201810522135 A CN 201810522135A CN 108849499 A CN108849499 A CN 108849499A
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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
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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
- A01H4/005—Methods for micropropagation; Vegetative plant propagation using cell or tissue culture techniques
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Abstract
The invention discloses a kind of methods for preparing Rhizoma Et Radix Notopterygii artificial seed, include the following steps：S1 is incubated for Rhizoma Et Radix Notopterygii somatic embryo in culture solution first；The preparation method of culture solution first：1.8~2.2mg 6-BA, 0.9~1.1mg NAA, 18~22g sucrose, 0.9~1.1g low-molecular weight chitoglycan, 0.9~1.1g oxalic acid and 36~44g sodium alginate are added in every liter of 1/10B5 culture medium；0.9~1.1g/100mLCaCl is added in the system that S2 obtains step S12In aqueous solution, it is incubated for, obtains Rhizoma Et Radix Notopterygii artificial seed.The present invention also protects a kind of culture solution for preparing Rhizoma Et Radix Notopterygii artificial seed, is the culture solution first.The efficient breeding of Rhizoma Et Radix Notopterygii seedling may be implemented in the present invention, meets the market demand of Rhizoma Et Radix Notopterygii seedling, has great promotional value.
Description
Technical field
The present invention relates to artificial seed Cultivating techniques, more specifically to a kind of method for preparing Rhizoma Et Radix Notopterygii artificial seed
And its special culture solution.
Background technique
Rhizoma Et Radix Notopterygii (Notopterygium Boissieu) belongs to Umbelliferae, celery subfamily, and delicious celery race is the endemic genus in China
One of, what wherein pharmacopeia was included has the kind such as Rhizoma Et Radix Notopterygii and notopterygium forbesii (river Qiang).The name of Rhizoma Et Radix Notopterygii is shown in《Sheng Nong's herbal classic》, be China most
One of early discovery and the drug of application, are had thousands of years of medicinal history, are used as medicine with rhizomes and root.
Rhizoma Et Radix Notopterygii has antibacterial, anti-inflammatory, analgesia, inhibits platelet aggregation and thrombosis, improve hemorheology, increase brain
Blood flow, antiviral and antiarrhythmic effect.Coronary disease and angina pectoris clinically mainly used for treating treats diarrhea, treatment
Colpomycosis, vulvitis treat dysmenorrhea, treat leucoderma, treat viral keratitis.
The resistance to shade of Rhizoma Et Radix Notopterygii, suitable for being grown in the high soil of the content of organic matter, cold resistant moist climate, therefore it is grown in height more
Mountain, Subalpine region shrubbery, thick grass and high mountain border, soil is based on subalpine shrub and meadow land soil, mountain forest soil, especially with soil
Loose, more containing humus and shady wet place distribution is more, is preferred in fertile acidity or neutral soil growth.Rhizoma Et Radix Notopterygii growth
It is regional stronger, should not be grown in temperature climate environment, Rhizoma Et Radix Notopterygii often with the associations such as various cuckoos and sallow.Growth cycle is
3~7 years, growing and cultivation was difficult, limits throughput.And the market demand sharply increases, Rhizoma Et Radix Notopterygii seed itself is few, the natural item in distributed areas
Part is severe, and natural propagation rate is very low, and weak tendency is obviously in Species Competition, and population is more rare, crosses in addition and adopts, disorderly adopts, is private
It is serious to adopt natural crude drugs, Rhizoma Et Radix Notopterygii ecological environment is caused seriously to be destroyed, Rhizoma Et Radix Notopterygii resource quantity is caused sharply to decline, it has also become is precious
Dilute endangered species, is included in《Chinese Plants Red Data Book》In, for national second level national key protected plant, influence based on Rhizoma Et Radix Notopterygii
Want the Chinese medicine production safety of raw material.
Summary of the invention
It is an object of the invention to develop a kind of method and its special culture solution for preparing Rhizoma Et Radix Notopterygii artificial seed, meet Rhizoma Et Radix Notopterygii
The market demand of seedling.
In order to achieve the above objectives, the present invention provides a kind of method and its special culture solution for preparing Rhizoma Et Radix Notopterygii artificial seed,
Include the following steps：S1, by 1.3 × 104~1.5 × 104A Rhizoma Et Radix Notopterygii somatic embryo is put into 1400~1600mL culture solution first simultaneously
It mixes, is stored at room temperature incubation 2~5 minutes；
The preparation method of the culture solution first：In every liter of 1/10 B5 medium be added 1.8~2.2mg, 6~BA, 0.9~
1.1mg NAA, 18~22g sucrose, 0.9~1.1g low-molecular weight chitoglycan, 0.9~1.1g oxalic acid and 36~44g sodium alginate；
The 1/10B5 culture medium：(NH4)2SO40.134g/L、KNO3 3g/L、NaH2PO4 0.15g/L、MgSO4·
7H2O 0.5g/L、CaCl2·2H2O 0.15g/L、MnSO4·4H2O 10g/L、ZnSO4·7H2O2g/L、H3BO33g/L、KI
0.75g/L、Na2MoO4·2H2O 0.25g/L、CuSO4·5H2O 0.025g/L、CoCl2·6H2O 0.025g/L、Na2-EDTA
3.725g/L、FeSO4·7H2O 2.785g/L, vitamin B1 1g/L, vitamin B6 0.1g/L, niacin 0.1g/L, inositol 1g/
L, sucrose 30g/L adds water to 1L；
Somatic embryo after S2, the incubation obtained with rubber head dropper aspiration step S1, and be added dropwise dropwise equipped with 2000~
0.5~1.5g/100mL of 4000mL CaCl2In the beaker of aqueous solution, it is stored at room temperature 20~40min of incubation, supernatant is abandoned, obtains
To in CaCl2Somatic embryo after being incubated in aqueous solution, then rinsed 3~4 times with aseptic deionized water, obtain artificial kind of Rhizoma Et Radix Notopterygii
Son.
Preferred embodiment is as follows：In the step S1, the condition of the incubation is：It is stored at room temperature incubation 3 minutes.
Preferred embodiment is as follows：In the step S2, the condition of the incubation is：It is stored at room temperature incubation 30 minutes.
Preferred embodiment is as follows：In the step S1, the preparation method of the culture solution first：Add in every liter of 1/10B5 culture medium
Enter 6~BA of 2mg, 1mg NAA, 20g sucrose, 1g low-molecular weight chitoglycan, 1g oxalic acid and 40g sodium alginate.
Preferred embodiment is as follows：In the step S1, the pH of the culture solution first is 6.0.
Preferred embodiment is as follows：CaCl in Rhizoma Et Radix Notopterygii somatic embryo, the culture solution first and the step S2 in the step S12
The proportion of aqueous solution is as follows：1.5×104A Rhizoma Et Radix Notopterygii somatic embryo：1500mL culture solution first：3000mL CaCl2Aqueous solution.
Preferred embodiment is as follows：Any description above prepares the method preparation gained Rhizoma Et Radix Notopterygii artificial seed of Rhizoma Et Radix Notopterygii artificial seed
Expanding propagation method：Include the following steps：
S21, the Rhizoma Et Radix Notopterygii artificial seed that step S2 is obtained is placed on B5 solid medium, 20~25 DEG C, intensity of illumination
2800~3000lx, 12 hours illumination/12 hour dark, 18~20d of alternation of light and darkness stationary culture；
S22, the Rhizoma Et Radix Notopterygii seedling after culture that step S21 is obtained is placed in air humidity 90%~95%, intensity of illumination
2800~3000lx, 20~25 DEG C, 12 hours illumination/12 hour dark are put under conditions of alternation of light and darkness and are cultivated in the medium
23~25d；
The B5 solid medium：50g agar is added in every liter of B5 fluid nutrient medium；
The B5 fluid nutrient medium：(NH4)2SO41.34g/L KNO330g/L, NaH2PO41.5g/L, MgSO4·
7H2O5g/L, CaCl2·2H2O1.5g/L, MnSO4·4H2O10g/L, ZnSO4·7H2O2g/L, H3BO33g/L, KI0.75g/L,
Na2MoO4·2H2O0.25g/L, CuSO4·5H2O0.025g/L, CoCl2·6H2O0.025g/L, Na2- EDTA3.725g/L,
FeSO4·7H2O2.785g/L, vitamin B1：1g/L vitamin B6：0.1g/L, niacin 0.1g/L, inositol 1g/L, sucrose 30g/
L adds water to 1L.
The step S22 used medium：Take sand, be rinsed with water, then mixed with 5g/100mL carbendazim aqueous solution it is wet,
It is rinsed with water after ten minutes 3 times.
The efficient breeding of Rhizoma Et Radix Notopterygii seedling may be implemented in the present invention, meets the market demand of Rhizoma Et Radix Notopterygii seedling, has great push away
Wide value.
Specific embodiment
Embodiment below facilitates a better understanding of the present invention, but does not limit the present invention.Experiment in following embodiments
Method is unless otherwise specified conventional method.Test material as used in the following examples is unless otherwise specified certainly
What routine biochemistry reagent shop was commercially available.Quantitative test in following embodiment is respectively provided with three repeated experiments, as a result makes even
Mean value.
The full name of 6~BA is 6~benzyl aminoadenine, purchased from the outstanding Bioisystech Co., Ltd in Shanghai three, catalog number
It is 1214~39~7.
The full name of NAA is α~methyl α-naphthyl acetate, and purchased from the outstanding Bioisystech Co., Ltd in Shanghai three, catalog number is 86~87
~3.
Low-molecular weight chitoglycan, molecular formula are " (C6H11NO4) n ", n=10~20, molecular weight is 2000~4000 dongles
, purchased from the outstanding Bioisystech Co., Ltd in Shanghai three, catalog number is 9012~76~4.
B5 Liquid Culture based formulas is shown in Table 1.
Table 1
B5 solid medium：50g agar is added in every liter of B5 fluid nutrient medium.
1/2B5 culture medium:(NH4)2SO4,0.67g/L、KNO3 7.5g/L、NaH2PO4 0.75g/L、MgSO4·7H2O
2.5g/L、CaCl2·2H2O 0.75g/L、MnSO4·4H2O 10g/L、ZnSO4·7H2O 2g/L、H3BO33g/L、KI
0.75g/L、Na2MoO4·2H2O 0.25g/L、CuSO4·5H2O 0.025g/L、CoCl2·6H2O 0.025g/L、Na2-EDTA
3.725g/L、FeSO4·7H2O 2.785g/L, vitamin B1 1g/L, vitamin B6 0.1g/L, niacin 0.1g/L, inositol 1g/
L, sucrose 30g/L, surplus are water.
1/10B5 culture medium：(NH4)2SO4,0.134g/L、KNO3 3g/L、NaH2PO4 0.15g/L、MgSO4·7H2O
0.5g/L、CaCl2·2H2O 0.15g/L、MnSO4·4H2O 10g/L、ZnSO4·7H2O 2g/L、H3BO33g/L、KI
0.75g/L、Na2MoO4·2H2O 0.25g/L、CuSO4·5H2O 0.025g/L、CoCl2·6H2O 0.025g/L、Na2-EDTA
3.725g/L、FeSO4·7H2O 2.785g/L, vitamin B1 1g/L, vitamin B6 0.1g/L, niacin 0.1g/L, inositol 1g/
L, sucrose 30g/L, surplus are water.
The preparation method of any description above Rhizoma Et Radix Notopterygii somatic embryo can be method first, include the following steps：
(I), the blade for taking Rhizoma Et Radix Notopterygii aseptic seedling, is cut into 0.5 × 0.5cm2, it is inoculated into culture medium first, 23 ± 2 DEG C of dark trainings
It supports 25 days；
(II), the embryo callus for taking step (I) to obtain is inoculated into the culture medium first, 23 ± 2 DEG C of dark culturings
25 days；
(III), the embryo callus for taking step (II) to obtain is inoculated into the culture medium first, 23 ± 2 DEG C of dark trainings
It supports 25 days；
(IV), the embryo callus that 1g step (III) obtains is added in 10ml culture medium second, 23 ± 2 DEG C, it is dark,
100rpm shaken cultivation 25 days；
(V), after completing step (IV), 1 parts by volume cultivating system is mixed with culture medium second described in 1 parts by volume, 23 ± 2
DEG C, it is dark, 100rpm shaken cultivation 25 days；
(VI), after completing step (V), 1 parts by volume cultivating system is mixed with culture medium second described in 1 parts by volume, 23 ± 2
DEG C, it is dark, 100rpm shaken cultivation 25 days；
(VII), after completing step (VI), 1 parts by volume cultivating system is mixed with culture medium second described in 1 parts by volume, 23 ± 2
DEG C, it is dark, 100rpm shaken cultivation 25 days；
(VIII) after completing step (VII), 1 parts by volume cultivating system is mixed with culture medium second described in 1 parts by volume, 23 ±
2 DEG C, dark, 100rpm shaken cultivation 25 days；
(IX) cultivating system that 1 parts by volume step (VIII) obtains is mixed with 2 parts by volume culture mediums third, 23 ± 2 DEG C, 12
Hour illumination/12 hour dark, 100rpm shaken cultivation 30 days；
The culture medium first is that the B5 of dichlorphenoxyacetic acid containing 0.5mg/L2,4-, 30g/L sucrose and 5g/L agar powder is cultivated
Base；The culture medium second is the 1/2B5 culture medium of dichlorphenoxyacetic acid containing 0.5mg/L2,4- and 30g/L sucrose；The culture medium
Third is the 1/2B5 culture medium containing 0.5mg/LNAA, 0.2mM TDZ, 0.4mg/L IBA, 0.4mg/L 6-BA and 30g/L sucrose.
In step (IX), the condition of the illumination can be 1500-2000lx, concretely 1500lx.
The preparation method of any description above Rhizoma Et Radix Notopterygii somatic embryo can be method second, include the following steps：
(1) embryo callus of Rhizoma Et Radix Notopterygii is added in culture medium second and is cultivated；The culture medium second is containing 0.5mg/
The 1/2B5 culture medium of L2,4- dichlorphenoxyacetic acid and 30g/L sucrose；
(2) after completing step (1), cultivating system is forwarded in culture medium third and is cultivated；The culture medium third be containing
1/2 B5 medium of 0.5mg/LNAA, 0.2mM TDZ, 0.4mg/LIBA, 0.4mg/L 6-BA and 30g/L sucrose.
In method second, the preparation method of the embryo callus of the Rhizoma Et Radix Notopterygii is as follows：The explant for taking Rhizoma Et Radix Notopterygii is inoculated into training
It supports on Ji Jia, is cultivated；The culture medium first is dichlorphenoxyacetic acid containing 0.5mg/L2,4-, 30g/L sucrose and 5g/L agar
The B5 medium of powder.
In method second, the preparation method of the embryo callus of the Rhizoma Et Radix Notopterygii is as follows：
1. taking the explant of Rhizoma Et Radix Notopterygii, it is inoculated into culture medium first, cultivates；
2. the embryo callus for taking step 1. to obtain is inoculated into the culture medium first, culture；
3. the embryo callus for taking step 2. to obtain is inoculated into the culture medium first, culture；
The culture medium first is that the B5 of dichlorphenoxyacetic acid containing 0.5mg/L2,4-, 30g/L sucrose and 5g/L agar powder is cultivated
Base.
The explant of the Rhizoma Et Radix Notopterygii is the leaf of Rhizoma Et Radix Notopterygii aseptic seedling or the stem of Rhizoma Et Radix Notopterygii aseptic seedling.The explant of the Rhizoma Et Radix Notopterygii is specific
It is as follows：The blade for taking Rhizoma Et Radix Notopterygii aseptic seedling, is cut into 0.5 × 0.5cm2.The explant of the Rhizoma Et Radix Notopterygii is specific as follows：Take Rhizoma Et Radix Notopterygii aseptic seedling
Stem, be cut into the stem section of 1cm.
1., 2. and 3. in, the condition of the culture is：23 ± 2 DEG C dark culturing 25 days.
The step (1) specifically comprises the following steps：
(1-1) adds to the embryo callus of 1g Rhizoma Et Radix Notopterygii in culture medium second described in 10ml, 23 ± 2 DEG C, dark, 100rpm
Shaken cultivation 25 days；
After (1-2) completes step (1-1), 1 parts by volume cultivating system is mixed with culture medium second described in 1 parts by volume, 23 ± 2
DEG C, it is dark, 100rpm shaken cultivation 25 days；
After (1-3) completes step (1-2), 1 parts by volume cultivating system is mixed with culture medium second described in 1 parts by volume, 23 ± 2
DEG C, it is dark, 100rpm shaken cultivation 25 days；
After (1-4) completes step (1-3), 1 parts by volume cultivating system is mixed with culture medium second described in 1 parts by volume, 23 ± 2
DEG C, it is dark, 100rpm shaken cultivation 25 days；
After (1-5) completes step (1-4), 1 parts by volume cultivating system is mixed with culture medium second described in 1 parts by volume, 23 ± 2
DEG C, it is dark, 100rpm shaken cultivation 25 days.
In the step (2), the condition of the culture is：23 ± 2 DEG C, 12 hours illumination/12 hour dark, shaken cultivations
30 days.
In the step (2), the proportion of cultivating system and the culture medium third is：
1 parts by volume cultivating system：2 parts by volume culture mediums third.
In the step (2), the shaken cultivation concretely 100rpm shaken cultivation.
In the step (2), the condition of the illumination can be 1500-2000lx, concretely 1500lx.
The invention also includes a kind of culture solutions for preparing Rhizoma Et Radix Notopterygii artificial seed, are culture solution first；The system of the culture solution first
Preparation Method：1.8-2.2mg 6-BA, 0.9-1.1mg NAA, 18-22g sucrose, 0.9-1.1g are added in every liter of 1/10B5 culture medium
Low-molecular weight chitoglycan, 0.9-1.1g oxalic acid and 36-44g sodium alginate.The preparation method of the culture solution first specifically may be used：Every liter
2mg 6-BA, 1mg NAA, 20g sucrose, 1g low-molecular weight chitoglycan, 1g oxalic acid and 40g alginic acid are added in 1/10B5 culture medium
Sodium.
Any description above low-molecular weight chitoglycan is the chitosan that molecular weight is 2000-4000 dalton.Any of the above
The molecular formula of the low-molecular weight chitoglycan is " (C6H11NO4) n ", n=10-20.Any description above low-molecular weight chitoglycan
Concretely purchased from the outstanding Bioisystech Co., Ltd in Shanghai three, catalog number is the chitosan of 9012-76-4.
Any description above 1/10B5 culture medium is concretely：NH4NO3 165mg/L、KNO3 190mg/L、KH2PO4
17mg/L、MgSO4·7H2O 37mg/L、CaCl2·2H2O 44mg/L、MnSO4·4H2O 22.3mg/L、ZnSO4·7H2O
8.6mg/L、H3BO36.2mg/L、KI 0.83mg/L、Na2MoO4·2H2O 0.25mg/L、CuSO4·5H2O 0.025mg/L、
CoCl2·6H2O 0.025mg/L、Na2-EDTA 37.25mg/L、FeSO4·7H2O 27.85mg/L, glycine 2mg/L, dimension life
Plain B10.4mg/L, vitamin B6 0.5mg/L, niacin 0.5mg/L, inositol 100mg/L, sucrose 30000mg/L, add water to 1L.
Embodiment 1 prepares somatic embryo
1, embryo callus is prepared
(1) blade for taking Rhizoma Et Radix Notopterygii aseptic seedling, is cut into 0.5 × 0.5cm2, it is inoculated into culture medium first, 20~25 DEG C of dark trainings
It supports 25 days.At this time it can be observed that growing light yellow callus, i.e. embryo callus from paddle cutout.
(2) embryo callus for taking step (1) to obtain, is inoculated into culture medium first, 20~25 DEG C dark culturing 25 days.
(3) embryo callus for taking step (2) to obtain, is inoculated into culture medium first, 20~25 DEG C dark culturing 25 days.
In practical operation, it can continue to carry out squamous subculture according to the method for step (2).
Culture medium first (solid)：Take B5 fluid nutrient medium, every liter of addition 0.5mg 2,4~dichlorphenoxyacetic acid, 30g sucrose
With 5g agar powder.
2, liquid suspension culture
(1) embryo callus that (3) of 1g step 1 obtain is added in 10ml culture medium second, 20~25 DEG C, it is dark,
100rpm shaken cultivation 25 days.
(2) after completing step (1), 1 parts by volume cultivating system is mixed with 1 parts by volume culture medium second, 20~25 DEG C, black
Secretly, 100rpm shaken cultivation 25 days.
(3) after completing step (2), 1 parts by volume cultivating system is mixed with 1 parts by volume culture medium second, 20~25 DEG C, black
Secretly, 100rpm shaken cultivation 25 days.
(4) after completing step (3), 1 parts by volume cultivating system is mixed with 1 parts by volume culture medium second, 20~25 DEG C, black
Secretly, 100rpm shaken cultivation 25 days.
(5) after completing step (4), 1 parts by volume cultivating system is mixed with 1 parts by volume culture medium second, 20~25 DEG C, black
Secretly, 100rpm shaken cultivation 25 days.
In practical operation, 3-5 squamous subculture can be carried out according to the method for step (2), obtain stable suspension cell
System.
Culture medium second (liquid)：Take 1/2B5 culture medium, every liter of addition 0.5mg 2,4- dichlorphenoxyacetic acid and 30g sucrose
's.
3, somatic embryo is prepared
The cultivating system that (5) of 1 parts by volume step 2 obtain is mixed with 2 parts by volume culture mediums third, 20~25 DEG C, 12 small
Shi Guangzhao (intensity of illumination 1500lx)/12 hours it is dark, 100rpm shaken cultivation 30 days, somatic embryo in cultivating system at this time
Concentration be more than or equal to 15000/L.
Culture medium third (liquid)：Take 1/2B5 culture medium, every liter of addition 0.5mg NAA (α~methyl α-naphthyl acetate), 0.2mmol TDZ
(thidiazuron), 0.4mg IBA (3~indolecarboxylic acid), 0.4mg 6-BA (6~benayl aminopurine) and 30g sucrose.
Embodiment 2 prepares Rhizoma Et Radix Notopterygii artificial seed according to the method provided by the invention
The preparation method (pH6.0) of culture solution first：Take 1/10B5 culture medium, every liter of 6~BA of addition 2mg, 1mg NAA,
20g sucrose, 1g low-molecular weight chitoglycan, 1g oxalic acid and 40g sodium alginate.
1, by 1.5 × 104The somatic embryo that a (about 10g) embodiment 1 obtains is put into 1500mL culture solution first and mixes,
It is stored at room temperature incubation 3 minutes.
2, the system that is obtained with rubber head dropper aspiration step 1 is simultaneously added dropwise dropwise equipped with 3000mL 1g/100mL CaCl2
It in the beaker of aqueous solution, is stored at room temperature and is incubated for 30min, abandon supernatant, rinsed 3~4 times with aseptic deionized water, obtain Rhizoma Et Radix Notopterygii people
Work post.
Embodiment 3 prepares Rhizoma Et Radix Notopterygii artificial seed according to existing method
The preparation method of culture solution penta：Take 1/2B5 culture medium, every liter of addition 30g sodium alginate.
The somatic embryo that embodiment 1 obtains is put into culture solution penta simultaneously 3~4min of soaking at room temperature, is then transferred to
0.5g/100mL CaCl2In aqueous solution and soaking at room temperature 3min, abandoning supernatant are rinsed 3~4 times with aseptic deionized water, are obtained
Rhizoma Et Radix Notopterygii artificial seed.
Embodiment 4, preparation Rhizoma Et Radix Notopterygii aseptic seedling
The Rhizoma Et Radix Notopterygii artificial seed of the Rhizoma Et Radix Notopterygii artificial seed for taking 100 embodiments 2 to prepare and the preparation of 100 embodiments 3, respectively
Carry out following steps：
1, Rhizoma Et Radix Notopterygii artificial seed is placed on B5 solid medium, 20~25 DEG C, the 12 hours illumination (light in the present embodiment
It is 3000lx according to intensity)/12 hours dark, stationary culture 20d (at this point, seedling that most of Seed Development bud is about 3cm).
2, after completing step 1, seedling is transplanted in seedling-growing container and (takes sand, is rinsed with water, then with the more bacterium of 5g/100mL
Lingshui Spring solution is mixed wet, is rinsed with water 3 times, is then divided in seedling-growing container after ten minutes), 20~25 DEG C, 12 hours illumination (this realities
Applying the intensity of illumination in example is 3000lx)/12 hours dark, air humidity 100%, stationary culture 25 days.
It is compared using method provided by the invention with existing method
Table 2
The seed amount sprouted when germination rate=completion step 1/seed total quantity × 100%.
The seed amount of seedling/seed total quantity × 100% when planting percent=completion step 2.
Seed total quantity=100.
It carries out repeating to test three times, results are averaged.
As shown in Table 2, big using the Rhizoma Et Radix Notopterygii artificial seed germination rate and planting percent of the method for the present invention preparation, existing method system
Standby Rhizoma Et Radix Notopterygii artificial seed germination rate and not of the invention big of planting percent, so the efficient of Rhizoma Et Radix Notopterygii seedling may be implemented in the present invention
Breeding, meets the market demand of Rhizoma Et Radix Notopterygii seedling, has great promotional value.
The foregoing is only a preferred embodiment of the present invention, but scope of protection of the present invention is not limited thereto,
Anyone skilled in the art within the technical scope of the present disclosure, according to the technique and scheme of the present invention and its
Inventive concept is subject to equivalent substitution or change, should be covered by the protection scope of the present invention.
Claims (7)
1. a kind of method for preparing Rhizoma Et Radix Notopterygii artificial seed, which is characterized in that include the following steps：
S1, by 1.3 × 104~1.5 × 104A Rhizoma Et Radix Notopterygii somatic embryo is put into 1400~1600mL culture solution first and mixes, room temperature
Stationary incubation 2~5 minutes；
The preparation method of the culture solution first：1.8~2.2mg, 6~BA, 0.9~1.1mg are added in every liter of 1/10B5 culture medium
NAA, 18~22g sucrose, 0.9~1.1g low-molecular weight chitoglycan, 0.9~1.1g oxalic acid and 36~44g sodium alginate；
1/10 B5 medium：(NH4)2SO40.134g/L、KNO3 3g/L、NaH2PO4 0.15g/L、MgSO4·7H2O
0.5g/L、CaCl2·2H2O 0.15g/L、MnSO4·4H2O 10g/L、ZnSO4·7H2O 2g/L、H3BO3 3g/L、KI
0.75g/L、Na2MoO4·2H2O 0.25g/L、CuSO4·5H2O 0.025g/L、CoCl2·6H2O 0.025g/L、Na2-EDTA
3.725g/L、FeSO4·7H2O 2.785g/L, vitamin B1 1g/L, vitamin B6 0.1g/L, niacin 0.1g/L, inositol 1g/
L, sucrose 30g/L adds water to 1L；
Somatic embryo after S2, the incubation obtained with rubber head dropper aspiration step S1, and be added dropwise dropwise equipped with 2000~
0.5~1.5g/100mL of 4000mL CaCl2In the beaker of aqueous solution, it is stored at room temperature 20~40min of incubation, supernatant is abandoned, obtains
To in CaCl2Somatic embryo after being incubated in aqueous solution, then rinsed 3~4 times with deionized water, obtain Rhizoma Et Radix Notopterygii artificial seed.
2. the method for preparation Rhizoma Et Radix Notopterygii artificial seed as described in claim 1, it is characterised in that：In the step S1, the incubation
Condition be：It is stored at room temperature incubation 3 minutes.
3. the method for preparation Rhizoma Et Radix Notopterygii artificial seed as described in claim 1, it is characterised in that：In the step S2, the incubation
Condition be：It is stored at room temperature incubation 30 minutes.
4. the method for preparation Rhizoma Et Radix Notopterygii artificial seed as described in claim 1, it is characterised in that：In the step S1, the culture
The preparation method of liquid first：It is poly- that 6~BA of 2mg, 1mg NAA, 20g sucrose, 1g low molecular weight shell are added in every liter of 1/10B5 culture medium
Sugar, 1g oxalic acid and 40g sodium alginate.
5. the method for preparation Rhizoma Et Radix Notopterygii artificial seed as described in claim 1, it is characterised in that：In the step S1, the culture
The pH of liquid first is 6.0.
6. the method for preparation Rhizoma Et Radix Notopterygii artificial seed as described in claim 1, it is characterised in that：Rhizoma Et Radix Notopterygii body cell in the step S1
CaCl in embryo, the culture solution first and the step S22The proportion of aqueous solution is as follows：1.5×104A Rhizoma Et Radix Notopterygii somatic embryo：
1500mL culture solution first：3000mL CaCl2Aqueous solution.
7. the expansion for preparing the method preparation gained Rhizoma Et Radix Notopterygii artificial seed of Rhizoma Et Radix Notopterygii artificial seed as described in claim 1~6 is any is numerous
Method, it is characterised in that：Include the following steps：
S21, the Rhizoma Et Radix Notopterygii artificial seed that step S2 is obtained is placed on B5 solid medium, 20~25 DEG C, intensity of illumination 2800~
3000lx, 12 hours illumination/12 hour dark, 18~20d of alternation of light and darkness stationary culture；
S22, the Rhizoma Et Radix Notopterygii seedling after culture that step S21 is obtained is placed in air humidity 90%~95%, intensity of illumination 2800~
3000lx, 20~25 DEG C, 12 hours illumination/12 hour dark, put under conditions of alternation of light and darkness culture 23 in the medium~
25d；
The B5 solid medium：50g agar is added in every liter of B5 fluid nutrient medium；
The B5 fluid nutrient medium：(NH4)2SO41.34g/L KNO330g/L, NaH2PO41.5g/L, MgSO4·7H2O5g/L,
CaCl2·2H2O1.5g/L, MnSO4·4H2O10g/L, ZnSO4·7H2O2g/L, H3BO33g/L, KI0.75g/L, Na2MoO4·
2H2O0.25g/L, CuSO4·5H2O0.025g/L, CoCl2·6H2O0.025g/L, Na2- EDTA3.725g/L, FeSO4·
7H2O2.785g/L, vitamin B1：1g/L vitamin B6：0.1g/L, niacin 0.1g/L, inositol 1g/L, sucrose 30g/L add water
To 1L.
The step S22 used medium：Take sand, be rinsed with water, then mixed with 5g/100mL carbendazim aqueous solution it is wet, 10 points
It is rinsed with water after clock 3 times.
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