CN109302987A - A kind of abductive approach of false-yellowflower milkwort root or herb callus - Google Patents
A kind of abductive approach of false-yellowflower milkwort root or herb callus Download PDFInfo
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- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
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Abstract
The invention discloses a kind of abductive approach of false-yellowflower milkwort root or herb callus.Abductive approach of the invention is using the non-germination seed of false-yellowflower milkwort root or herb select tree as explant, obtain the culture medium prescription that optimum seed sprouts culture and aseptic seedling Multiplying culture, the culture medium prescription of suitable sterile seedling stem section and Callus of Leaf induction is filtered out, scientific basis is provided for the effective secondary metabolism ingredient of substitution Source Study and cell suspension cultures acquisition of false-yellowflower milkwort root or herb plant resources, is laid a good foundation for subsequent industrialization development research.
Description
Technical field
The invention belongs to field of plant growing technology, and in particular to a kind of abductive approach of false-yellowflower milkwort root or herb callus.
Background technique
False-yellowflower milkwort root or herb (Polygala fallax Hemsl) belongs to Polygalaceae (Polygalaceae) rogation flower
(Polygala) machaka is distributed mainly on the ground such as Hunan, Guangxi, Guangdong, Fujian and Yunnan, is a kind of time-honored people
Between drug, sweet in flavor with root or all herbal medicine, slight bitter is mild-natured.With blood-enrich, invigorating spleen to remove dampness, activating microcirculation and removing stasis medicinal and other effects is faced
Physically weak after being ill, soreness of waist and knee joint, traumatic injury, the treatment of edema due to deficiency of the spleen, irregular menstruation etc. are used on bed.False-yellowflower milkwort root or herb mainly contains
There is the various actives substance such as saponin(e, polysaccharide, ketone, organic acid and amino acid, exempts from modern pharmacological studies have shown that it has to adjust
The effects of epidemic disease, resisting stress, anti-aging, anti-oxidant, blood activating and fat reducing, has and compare broad application prospect and value of exploiting and utilizing.It is yellow
Flower omei meadowrue herb artificial growth is mainly based on seed and cutting propagation.But seed production is low, and picking time is short, and harvesting is difficult；And it is wild
Production-goods source is limited, seed collecting resource critical shortage；Though vegetative propagation energy fast propagating seedling wood, and its merit is kept, chrysanthemum pours
Lotus growth is slow, and plant branch is few, cutting propagation limited material；And tissue-cultured seedling reproductive-cost is high, transplanting survival rate is low, growth cycle
It is long.
Summary of the invention
It is an object of the invention to overcome prior art defect, a kind of induction side of false-yellowflower milkwort root or herb callus is provided
Method.
Technical scheme is as follows:
A kind of abductive approach of false-yellowflower milkwort root or herb callus, includes the following steps:
(1) explant selects: using the non-germination seed of false-yellowflower milkwort root or herb select tree as explant, cleaning to the explant
And disinfection；
(2) sprout culture: the explant after step (1) is cleaned and sterilized is placed in germination medium, in dark item
It carries out sprouting culture 29-32d for 25 ± 2 DEG C under part, obtains aseptic seedling, which is added based on MS minimal medium
Add sucrose 24-26g/L, agar 6.8-7.2g/L, 6-BA 0.95-1.05mg/L, NAA 0.48-0.51mg/L, pH=5.75-
5.86；
(3) Multiplying culture: cutting the stem-segment with node of a length of 1~2cm of the aseptic seedling of step (1) acquisition, is placed in proliferation training
It supports and carries out Multiplying culture 29-32d in base, form more Multiple Buds in aseptic seedling to induce, the proliferated culture medium is basic with MS
Based on culture medium, sucrose 24-26g/L, agar 6.8-7.2g/L, 6-BA 0.95-1.05mg/L, NAA 0.19- are added
0.21mg/L, pH=5.75-5.86；The condition of above-mentioned Multiplying culture are as follows: intensity of illumination 1200-2000LuX, light application time
12-16h/d, temperature are 25 ± 2 DEG C；
(4) aseptic seedling through step (3) Multiplying culture callus from stem segment Fiber differentiation: is cut into 0.5~1cm segment
Afterwards, it accesses and carries out Fiber differentiation 29-32d in stem section calli induction media, to obtain callus from stem segment, which is lured
Culture medium is led based on B5 medium, adds sucrose 38-42g/L, agar 5.8-6.2g/L, 6-BA 0.48-0.53mg/L,
NAA 3.9-4.2mg/L, pH=5.75-5.86；The condition of above-mentioned callus from stem segment Fiber differentiation are as follows: intensity of illumination is
1200-2000LuX, light application time 8-12h/d, temperature are 25 ± 2 DEG C.
In a preferred embodiment of the invention, the germination medium is based on MS minimal medium, addition
Sucrose 25g/L, agar 7g/L, 6-BA 1mg/L, NAA 0.5mg/L, pH=5.8.
In a preferred embodiment of the invention, the proliferated culture medium is based on MS minimal medium, addition
Sucrose 25g/L, agar 7g/L, 6-BA 1mg/L, NAA 0.2mg/L, pH=5.8.
In a preferred embodiment of the invention, the stem section calli induction media is based on B5 medium,
Add sucrose 40g/L, agar 6g/L, 6-BA 0.5mg/L, NAA 4mg/L, pH=5.8.
Another technical solution of the invention is as follows:
A kind of abductive approach of false-yellowflower milkwort root or herb callus, includes the following steps:
(1) explant selects: using the non-germination seed of false-yellowflower milkwort root or herb select tree as explant, cleaning to the explant
And disinfection；
(2) sprout culture: the explant after step (1) is cleaned and sterilized is placed in germination medium, in dark item
It carries out sprouting culture 29-32d for 25 ± 2 DEG C under part, obtains aseptic seedling, which is added based on MS minimal medium
Add sucrose 24-26g/L, agar 6.8-7.2g/L, 6-BA 0.95-1.05mg/L, NAA 0.48-0.51mg/L, pH=5.75-
5.86；
(3) miaoye for the aseptic seedling that step (2) obtain Callus of Leaf Fiber differentiation: is cut into 0.48-0.51cm*
After the small cube of 0.48-0.51cm, accesses and carry out Fiber differentiation 29-32d in blade calli induction media, be cured with obtaining blade
Injured tissue, the blade calli induction media add sucrose 24-26g/L, agar 5.8-6.2g/L based on B5 medium,
2,4-D 3.8-4.1mg/L, NAA 0.48-0.53mg/L, pH=5.75-5.86；Above-mentioned callus from stem segment Fiber differentiation
Condition are as follows: intensity of illumination 1200-2000LuX, light application time 8-12h/d, temperature are 25 ± 2 DEG C.
In a preferred embodiment of the invention, the germination medium is based on MS minimal medium, addition
Sucrose 25g/L, agar 7g/L, 6-BA 1mg/L, NAA 0.5mg/L, pH=5.8.
In a preferred embodiment of the invention, the blade calli induction media is based on B5 medium,
Add sucrose 25g/L, agar 6g/L, 2,4-D 4mg/L, NAA 0.5mg/L, pH=5.8.
The beneficial effects of the present invention are: abductive approach of the invention is using the non-germination seed of false-yellowflower milkwort root or herb select tree as explant
Body obtains the culture medium prescription that optimum seed sprouts culture and aseptic seedling Multiplying culture, has filtered out suitable sterile seedling stem
The Fiber differentiation based formulas of section and Callus of Leaf induction is the substitution Source Study and cell of false-yellowflower milkwort root or herb plant resources
The culture that suspends obtains effective secondary metabolism ingredient and provides scientific basis, lays a good foundation for subsequent industrialization development research.
Detailed description of the invention
Fig. 1 is the photo of the sprouting of seed in the embodiment of the present invention 1.
Fig. 2 is the photo that callus is formed when seed is sprouted in the embodiment of the present invention 1.
Fig. 3 is one of the photo of aseptic seedling that seed is sprouted in the embodiment of the present invention 1.
Fig. 4 is the two of the photo for the aseptic seedling that seed is sprouted in the embodiment of the present invention 1.
Fig. 5 is one of the photo of Multiplying culture of stem section evoking adventive bud in the embodiment of the present invention 1.
Fig. 6 is two of the photo of the Multiplying culture of stem section evoking adventive bud in the embodiment of the present invention 1.
Fig. 7 is the photo of the callus of stem section induction in the embodiment of the present invention 1.
Fig. 8 is the photo of the callus of stem section induction in the embodiment of the present invention 1.
Fig. 9 is the photo of the callus of blade induction in the embodiment of the present invention 1.
Figure 10 is the photo of the squamous subculture of callus from stem segment in the embodiment of the present invention 1.
Specific embodiment
Technical solution of the present invention is further explained and described below by way of specific embodiment.
In following embodiments,
MS culture medium prescription:
1650mg/L NH4NO3、1900mg/L KNO3、440mg/L CaCl2·2H2O、370mg/LMgSO4·7H2O、
170mg/L KH2PO4、0.83mg/L KI、6.2mg/L H3BO3、0.25mg/L Na2MoO4·2H2O、22.3mg/L MnSO4·
H2O、8.6mg/L ZnSO4·7H2O、0.025mg/L CuSO4·5H2O、0.025mg/L CoCl2·6H2O、227.8mg/L
FeSO4·7H2O、37.3mg/L Na2EDTA, 100mg/L inositol, 0.1mg/L vitamin B1, 0.5mg/L niacin, 0.5mg/L dimension
Raw element B6, 2.0mg/L glycine.
B5 medium formula: 2500mg/L KNO3、150mg/L CaCl2·2H2O、250mg/LMgSO4·7H2O、
134mg/L(NH4)2SO4、150mg/L NaH2PO4·H2O、3mg/L H3BO3、0.75mg/L KI、0.25mg/L Na2MoO4·
2H2O、10mg/L MnSO4·H2O、2mg/L ZnSO4·7H2O、0.025mg/L CuSO4·5H2O、0.025mg/L CoCl2·
6H2O、27.8mg/L FeSO4·7H2O、37.3mg/L Na2MoEDTA, 100mg/L inositol, 10mg/L vitamin B1, 1mg/L cigarette
Acid, 1mg/L vitamin B6。
Embodiment 1
1.1 test material
Using the non-germination seed of Zhangzhou City of Fujian Province Nanjing County false-yellowflower milkwort root or herb select tree as test material.
1.2 test method
1.2.1 culture is sprouted
The acquisition and disinfection of explant: this experiment is using the non-germination seed of false-yellowflower milkwort root or herb select tree as explant, first flowing water
1h is rinsed, then impregnates 0.5h with dish washing liquid dilute solution, then wiped with cotton, is finally used distilled water flushing 3 times.Drain away the water shifting
Enter progress surface sterilization processing in superclean bench: after first vibrating flushing material 60s with 70% alcohol, with sterile water wash one
Time, then vibrated with 0.I%HClO and impregnate 20min, aseptic water washing 3~5 times, about 2min every time.With MS minimal medium, sucrose
25g/L, agar 7g/L, pH5.8, add different hormone combinations (being shown in Table 1) respectively, and 30 explants of each processing are repeated 3 times；
In the case where temperature is 25 ± 2 DEG C, statistics, germination rate (%)=(the explant number of rudiment/explant sum * are observed after dark culture 30d
100%), and Aseptic Seedling Growth situation is observed.
1 false-yellowflower milkwort root or herb seed of table sprouts the hormone combinations of culture
1.2.2 Multiplying culture
The stem-segment with node for sprouting a length of 1-2cm of aseptic seedling that culture obtains is cut, the culture of aseptic seedling shoot proliferation is carried out, with
More Multiple Buds are induced to be formed.Using MS as minimal medium, 6-BA (0.2,0.5,1) mg/L+NAA0.2mg/L, sucrose are added
25g/L, agar 7g/L, pH5.8 study inductive effect of the concentration proportioning to false-yellowflower milkwort root or herb Multiple Buds of hormon.Each
20 explants are handled, are repeated 3 times.Seedling and bud proliferation multiple (proliferation times=proliferation sprout number/inoculation number) are counted after 30d, and
Observe and record each processing sprout growing state.
1.2.3 hormone combines the influence to the induction of false-yellowflower milkwort root or herb stem section callus with sucrose various concentration
Callus induction research is carried out as material using the aseptic seedling obtained in 1.2.2 Multiplying culture.In vitro culture is obtained
Aseptic seedling is cut into 0.5-1cm segment and is respectively connected in different induced mediums.Using L9(34) Orthogonal Experiment and Design, choose 6-
BA (0,0.5,1mg/L), NAA (1,2,4mg/L), sucrose concentration (20,30,40mg/L) is Three factors-levels, with B5For base
Basal culture medium, agar 6g/L, pH5.8, totally 9 processing (being shown in Table 4), 20 explants of each processing are repeated 3 times, after cultivating 30d
Stem section Callus induction rate (the explant number/explant sum for forming callus) is counted, and observes callus upgrowth situation.
1.2.4 the influence that hormone concentration and carbon source various combination induce false-yellowflower milkwort root or herb blade callus
It cuts 1.2.1 and sprouts the aseptic seed seedling leaf progress callus induction obtained in culture.By aseptic seed seedling
Blade on plant is cut into the small cube of 0.5cm*0.5cm, is respectively connected to the B5 medium of additional hormone and carbon source various combination
In, using L9(34) Orthogonal Experiment and Design, with NAA (0.1,0.5,1mg/L), 2,4-D (1,2,4mg/L), carbon source kind (sucrose,
Glucose, white granulated sugar) it is experimental factor, carbon source concentration is 25g/L, agar 6g/L, pH5.8, totally 9 processing (being shown in Table 5), often
20 explants of a processing, are repeated 3 times, and count Callus induction rate (%)=(explant number of formation callus/outer after cultivating 30d
Implant sum) * 100%, observe callus upgrowth situation.
1.2.5 condition of culture
Above-mentioned seed is cultivated after sprouting, the intensity of illumination of aseptic seedling Multiplying culture is 1200-2000LuX, light application time 12-
16h/d, temperature are 25 ± 2 DEG C；The intensity of illumination of callus Fiber differentiation is 1200-2000LuX, light application time 8-12h/d, temperature
It is 25 ± 2 DEG C.
1.2.5 data statistics and analysis
Data are handled using Excel2003 software and SPSS Statistics 17.0.It is shown with Duncan method
The analysis of work property.
2 results and analysis
The screening of 2.1 germination mediums
Influence of the different hormone combinations processing to false-yellowflower milkwort root or herb seed asepsis sprouting is different (being shown in Table 2).Wherein at Z1, Z2
It is thin and delicate to manage aseptic seedling, slow growth (see Fig. 3)；Z3 processing seed embryo broken skin is directly formed callus (see Fig. 2), at Z4, Z5
Reason seed starts to sprout (see Fig. 1) after two weeks, and late growth is more vigorous, and robust plant, blade is more, well developed root system (see Fig. 4).
The aseptic seedling that Z6 processing is sprouted is thinner and more delicate, some sprouts a small amount of callus occur in epicotyl base portion.
Influence of 2 different hormone combinations of table to false-yellowflower milkwort root or herb seed germination and growth
Note: capitalization indicates the otherness in 0.01 level in same row, and lowercase indicates in 0.05 level
Otherness (similarly hereinafter).
As seen from the results in Table 5, hormon has reached extremely significant water with the influence for comparing false-yellowflower milkwort root or herb seed germination rate
Flat (F=74.71 > F0.01 (5,12)=5.06) seed germination rate highest, is handled with Z5, is taken second place for 76.7%, Z4 processing, the two
Difference is extremely significant；With the raising of NAA concentration, the decline of false-yellowflower milkwort root or herb seed germination rate.Find there is addition 2,4-D in test
Culture medium combination be unfavorable for the sprouting of false-yellowflower milkwort root or herb seed, seed germination rate is relatively low, handles highest with Z2, only
33.3%；And add in the culture medium combination of NAA, false-yellowflower milkwort root or herb seed germination rate is higher, 50% or more；Low concentration
NAA is conducive to the growth that seed sprouts aseptic seedling, and plant is more healthy and stronger, and Aseptic Seedling Growth in the culture medium of the NAA containing higher concentration
Thinner and more delicate, seed germination rate reduces.For this purpose, selecting Z5 processing combination: MS+1mg/L 6-BA+0.5mg/L NAA is chrysanthemum pouring
Lotus seed sprouts optimum culture medium prescription.
The screening of 2.2 proliferated culture mediums
Stem-segment with node is inoculated into subculture medium, different subculture mediums are to false-yellowflower milkwort root or herb clump bud seedling shoot proliferation
Effect is different.Most of stem section grows sprouting or differentiates callus after 10-15d, and statistical observation is found after 30d, is trained with H3
It supports base and combines seedling and bud proliferation coefficient highest, be 4.7, callus is obvious, but sprout plant is thinner and more delicate, and partial blade is rolled up
There is a small amount of vitrifying seedling in bent (see Fig. 5), Later growth；The clump bud seedling robust growth of H1 and H2 culture medium combination induction, blade
Greatly, but growth coefficient is lower, respectively 2.4 and 3.5, wherein finds stem section base portion with a small amount of green in the combination of H2 culture medium
Color callus (see Fig. 6).
3 false-yellowflower milkwort root or herb stem section adventitious bud inducing Multiplying culture effect of table
Result is analyzed by table 3 it is found that the 6-BA of various concentration is to false-yellowflower milkwort root or herb stem section adventitious bud inducing growth coefficient
Influence reaches extremely significant level (F=61.569 > F0.01 (2,6)=10.92).With the increase of 6-BA concentration, false-yellowflower milkwort root or herb stem
Section adventitious bud inducing growth coefficient improves；When 6-BA concentration is 2mg/L, false-yellowflower milkwort root or herb stem section adventitious bud inducing growth coefficient
Highest is 4.7, when being respectively 0.5mg/L and 1mg/L with 6-BA concentration, the growth coefficient of false-yellowflower milkwort root or herb stem section adventitious bud
Significant difference, but after adventitious bud proliferation difference of coefficients between the two it is not significant.For this purpose, selection H2 processing: MS+1mg/L 6-BA+
0.2mg/L is the optimum medium of false-yellowflower milkwort root or herb stem section adventitious bud inducing squamous subculture.
2.3 hormones combine the influence to the induction of false-yellowflower milkwort root or herb stem section callus with sucrose various concentration
Stem section is accessed in 9 groups of different culture mediums and carries out callus induction, each processing is in the 15-20d time-division
Callus is dissolved, each callus divergaence time that handles is not much different.Most of processing forms callus in internode or wound
Tissue, part stem section also differentiate clump bud.The callus growth situation that various combination processing stem section induces is different.J1,J6,
The callus amount that J8 processing group stem section differentiates is less, and quality has a small amount of buds differentiation compared with consolidation；J2, J4, J9 processing group stem
The callus amount of section induction is relatively more, and quality slightly consolidation, some is more bulk, and especially closing in J4, J9 processing group has individually
Sprout is differentiated in the callus of consolidation (see Fig. 7).The callus quality of J3, J5, J7 processing combination stem section induction is slightly bulk, soft,
Mostly yellow green, rare buds differentiation in callus, Callus induction rate is higher, and callus amount is more (see Fig. 8)；Wherein J5, J7
The callus color of induced synthesis is wet, grows in later period several times callus squamous subculture and stablizes, breeds fast (see Figure 10).
4 hormone of table combines the influence to the induction of false-yellowflower milkwort root or herb callus from stem segment with sucrose concentration
Note: K represents the average value of each horizontal inductivity of each factor, and R indicates that each three levels of factor averagely induce
Very poor (similarly hereinafter) of rate
By 4 test result of table it is found that influence of the different disposal to Callus induction rate has reached extremely significant level (F=
37.434 > F0.01 (8,18)=3.71).Each very poor size analysis of factor level, can obtain 3 factors to stem section callus from table 4
The primary and secondary sequence for organizing inductivity to influence are as follows: NAA > 6-BA > sucrose concentration.It is averaged in terms of inductivity from single factor, with NAA
Concentration increases, and callus induction rate improves, and illustrates that the NAA within the scope of a certain concentration is conducive to the induction of callus from stem segment.
Combination Callus induction rate highest is wherein handled with J3, is 93.3%, but to combine difference not significant with J5 processing.In view of stem section lures
Difficulty or ease of the callus led in later period squamous subculture, selecting J5 processing combination is B5+0.5mg/L6-BA+4mg/LNAA+
40g/L sucrose is that false-yellowflower milkwort root or herb stem section callus induces most suitable culture medium prescription.
The influence that 2.4 hormones and carbon source various combination induce false-yellowflower milkwort root or herb blade callus
False-yellowflower milkwort root or herb blade is cut into culture medium of the access of the rectangular fritter with wound containing hormone Yu carbon source various combination
In, observation finds that most of leaf chlorosis turns yellow after two weeks for inoculation, and a small amount of culture callus occurs in the wound of blade.At this
It is found in research, the more difficult induced synthesis callus of false-yellowflower milkwort root or herb blade, different culture medium group is closed, and inducer blade formation is cured
The case where injured tissue difference.The amount that Y1, Y6, Y8 culture medium combine inducer blade formation callus is more, remaining processing
The amount for combining inducer blade formation callus is less.Wherein the Callus of Leaf color of Y2, Y6, Y8 culture medium combination induction is green
Color, wet, Y2, Y5 callus quality consolidation, the callus quality of Y1 induction is loose, yellow green, Y6, Y8 callus matter
Ground is relatively soft, green (see Fig. 9).The Callus of Leaf of each processing induction is showed no the differentiation of sprout, induces shape with Y6, Y8
At soft, green, the wet callus of quality can grow stabilization in 3 times squamous subculture, remaining handle then transfer after
Grow unstable after generation, variable xanthochromia is brown.
The influence that 5 hormone of table and carbon source combination induce false-yellowflower milkwort root or herb Callus of Leaf
Result is analyzed by table 5 it is found that hormone and carbon source various combination are to false-yellowflower milkwort root or herb Callus of Leaf inductivity
Influence extremely significant (F=5.595 > F0.01 (8,18)=3.71), horizontal very poor size analysis can obtain three out of each in table 5 factor
The primary and secondary sequence that a factor influences Callus of Leaf inductivity are as follows: 2,4-D > NAA > carbon sources.Contain as can be seen from the table
The medium treatment of high concentration NAA combines, and Callus of Leaf inductivity is higher, handles Callus induction rate highest with Y1, is
28.3%, followed by Y8 processing, Callus of Leaf inductivity is 24%, but the difference between the two is not significant.Remaining processing blade is cured
Injured tissue inductivity is below 20%.Comprehensively consider, selecting Y8 processing is B5+0.5mg/LNAA+4mg/L2,4-D+25g/L
Sucrose is the optimum medium formula of inducer blade callus.
3 conclusions
In conclusion being accessed in germination medium after false-yellowflower milkwort root or herb seed is preprocessed and disinfection treatment: the punching of first flowing water
1h is washed, dish washing liquid dilute solution impregnates 0.5h, then is wiped with cotton, finally uses distilled water flushing 3 times；Draining away the water, it is ultra-clean to move into
In workbench, 70% ethyl alcohol vibrate 60s, rinsed with sterile water one time, then with 0.1%HClO vibrate impregnate 20min, rinsed with sterile water
3~5 times.The optimum culture medium group of false-yellowflower milkwort root or herb seed asepsis sprouting is combined into MS+1mg/L 6-BA+0.5mg/L NAA+ sugarcane
Sugared 25g/L+7g/L agar, the optimum medium of false-yellowflower milkwort root or herb stem section adventitious bud inducing shoot proliferation are MS+1mg/L 6-BA+
0.2mg/L+ sucrose 25g/L+7g/L agar；The optimum culture medium group of false-yellowflower milkwort root or herb callus from stem segment induction is combined into B5+
0.5mg/L6-BA+4mg/LNAA+40g/L sucrose+6g/L agar；Callus of Leaf induces most suitable culture group to be combined into B5+
0.5mg/LNAA+4mg/L2,4-D+25g/L sucrose+6g/L agar are the optimum medium formula of inducer blade callus.With
Appropriate materials of the false-yellowflower milkwort root or herb stem section as callus induction.
The foregoing is only a preferred embodiment of the present invention, the range that the present invention that therefore, it cannot be limited according to is implemented, i.e.,
Equivalent changes and modifications made in accordance with the scope of the invention and the contents of the specification should still be within the scope of the present invention.
Claims (7)
1. a kind of abductive approach of false-yellowflower milkwort root or herb callus, characterized by the following steps:
(1) explant selects: using the non-germination seed of false-yellowflower milkwort root or herb select tree as explant, which being cleaned and is disappeared
Poison；
(2) sprout culture: the explant after step (1) is cleaned and sterilized is placed in germination medium, under dark condition
25 ± 2 DEG C carry out sprouting culture 29-32d, obtain aseptic seedling, which adds sugarcane based on MS minimal medium
Sugared 24-26g/L, agar 6.8-7.2g/L, 6-BA 0.95-1.05mg/L, NAA 0.48-0.51mg/L, pH=5.75-5.86；
(3) Multiplying culture: the stem-segment with node of a length of 1~2cm of the aseptic seedling of step (1) acquisition is cut, proliferated culture medium is placed in
Middle progress Multiplying culture 29-32d, forms more Multiple Buds to induce, which is cultivated substantially with MS in aseptic seedling
Based on base, sucrose 24-26g/L, agar 6.8-7.2g/L, 6-BA 0.95-1.05mg/L, NAA 0.19-0.21mg/ are added
L, pH=5.75-5.86；The condition of above-mentioned Multiplying culture are as follows: intensity of illumination 1200-2000LuX, light application time 12-16h/d,
Temperature is 25 ± 2 DEG C；
(4) it callus from stem segment Fiber differentiation: by after the aseptic seedling of step (3) Multiplying culture is cut into 0.5~1cm segment, connects
Enter progress Fiber differentiation 29-32d in stem section calli induction media, to obtain callus from stem segment, stem section callus induction training
Base is supported based on B5 medium, adds sucrose 38-42g/L, agar 5.8-6.2g/L, 6-BA 0.48-0.53mg/L, NAA
3.9-4.2mg/L, pH=5.75-5.86；The condition of above-mentioned callus from stem segment Fiber differentiation are as follows: intensity of illumination 1200-
2000LuX, light application time 8-12h/d, temperature are 25 ± 2 DEG C.
2. abductive approach as described in claim 1, it is characterised in that: the germination medium is using MS minimal medium as base
Plinth adds sucrose 25g/L, agar 7g/L, 6-BA 1mg/L, NAA 0.5mg/L, pH=5.8.
3. abductive approach as described in claim 1, it is characterised in that: the proliferated culture medium is using MS minimal medium as base
Plinth adds sucrose 25g/L, agar 7g/L, 6-BA 1mg/L, NAA 0.2mg/L, pH=5.8.
4. abductive approach as described in claim 1, it is characterised in that: the stem section calli induction media is with B5 medium
Sucrose 40g/L, agar 6g/L, 6-BA 0.5mg/L, NAA 4mg/L, pH=5.8 are added in basis.
5. a kind of abductive approach of false-yellowflower milkwort root or herb callus, characterized by the following steps:
(1) explant selects: using the non-germination seed of false-yellowflower milkwort root or herb select tree as explant, which being cleaned and is disappeared
Poison；
(2) sprout culture: the explant after step (1) is cleaned and sterilized is placed in germination medium, under dark condition
25 ± 2 DEG C carry out sprouting culture 29-32d, obtain aseptic seedling, which adds sugarcane based on MS minimal medium
Sugared 24-26g/L, agar 6.8-7.2g/L, 6-BA 0.95-1.05mg/L, NAA 0.48-0.51mg/L, pH=5.75-5.86；
(3) miaoye for the aseptic seedling that step (2) obtain Callus of Leaf Fiber differentiation: is cut into 0.48-0.51cm*0.48-
After the small cube of 0.51cm, accesses and carry out Fiber differentiation 29-32d in blade calli induction media, to obtain blade callus group
It knits, which adds sucrose 24-26g/L, agar 5.8-6.2g/L, 2,4-D based on B5 medium
3.8-4.1mg/L NAA 0.48-0.53mg/L, pH=5.75-5.86；The condition of above-mentioned callus from stem segment Fiber differentiation are as follows:
Intensity of illumination is 1200-2000LuX, and light application time 8-12h/d, temperature is 25 ± 2 DEG C.
6. abductive approach as claimed in claim 5, it is characterised in that: the germination medium is using MS minimal medium as base
Plinth adds sucrose 25g/L, agar 7g/L, 6-BA 1mg/L, NAA 0.5mg/L, pH=5.8.
7. abductive approach as claimed in claim 5, it is characterised in that: the blade calli induction media is with B5 medium
Sucrose 25g/L, agar 6g/L, 2,4-D 4mg/L, NAA 0.5mg/L, pH=5.8 are added in basis.
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