CN104885937B - A kind of method for inducing tuniclike psammosilene root callus to produce anthocyanidin - Google Patents
A kind of method for inducing tuniclike psammosilene root callus to produce anthocyanidin Download PDFInfo
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- CN104885937B CN104885937B CN201510247199.2A CN201510247199A CN104885937B CN 104885937 B CN104885937 B CN 104885937B CN 201510247199 A CN201510247199 A CN 201510247199A CN 104885937 B CN104885937 B CN 104885937B
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Abstract
The invention discloses a kind of method for inducing tuniclike psammosilene root callus to produce anthocyanidin, it is that to select the young tender shoots of tuniclike psammosilene root aseptic seedling be explant, in MS minimal mediums, evoked callus under conditions of the additional mg/L of the 6 BA 0.5 and mg/L of IAA 2.5.The white loose callus for producing will be induced to be placed on MS minimal mediums, the inducible anthocyanidin of culture is produced under the conditions of the addition mg/L of the 6 BA 0.5 and mg/L of NAA 1.5, the lx of illumination 2000.To induce again and generate the callus of anthocyanidin and carry out shoot proliferation culture, with MS as minimal medium, 6 BA of addition 1.0 mg/L, NAA 0.5 mg/L and mg/L of KT 0.3 will be final to obtain anthocyanidin content callus higher.The anthocyanidin that present invention induction is produced is a kind of healthy nutritive value pure natural pigment high, beautiful in colour.
Description
Technical field
The present invention relates to field of plant tissue culture technique, and in particular to tuniclike psammosilene root callus induction and tuniclike psammosilene root callus
The regulation and control method that tissue anthocyanidin is formed.
Background technology
Tuniclike psammosilene root（Psammosilene tunicoides W.C.Wu et C.Y.Wu）Also known as RADIX PSAMMOSILENE, Sinense Knotweed Rhizome etc.,
Belong to Caryophyllaceae tuniclike psammosilene root and belong to perennial herb, be distributed mainly on the ground such as China Guizhou, Yunnan, Sichuan, Tibet, be grown in height above sea level
In the gravel hillside of 2000~3800 m or calcium carbonate rock seam.Tuniclike psammosilene root is a kind of Endangered Medicinal Herb, and its root is used as medicine, has
The functions such as scattered silt analgesic, toxin expelling hemostasis, are put into for 1991 as rare endangered species《Chinese Plants Red Data Book》First,
It is put into as Chinese Second Class Key Protected Plant within 1999《National key protected wild plants register（First）》In.Tuniclike psammosilene root exists
Yunnan Medicinal history is more long, and the Chinese patent medicine preparation developed using modern means of science and technology has longstalk condorvine herb to dissipate, fracture and pain-stop cream, gold
Bone lotus capsule etc..In purple green more than tuniclike psammosilene root plants stems, duration of flowering petal is aubergine, i.e. the cell of tuniclike psammosilene root plant has conjunction
Into the ability of natural pigment, therefore, while using its root, the natural pigment of the comprehensive exploitation species also has a extensive future.
The content of the invention
The invention provides a kind of method for inducing tuniclike psammosilene root callus to produce anthocyanidin, the day needed for enriching people
The species of right phytochrome, also for the comprehensive development and utilization of tuniclike psammosilene root this rare resources opens new way.
The technical solution adopted by the present invention：Callus first is produced by explant induction of tuniclike psammosilene root aseptic seedling, then is passed through
To the regulation of plant growth regulating substance concentration and intensity of illumination with optimize callus anthocyanidin formation condition.
Comprise the following steps that：
Step 1：Using tuniclike psammosilene root aseptic seedling as explant, it is placed in MS culture mediums, adds the mg/L of 6-BA 0~3.0 and IAA
0~2.0 mg/L is cultivated, and induction obtains callus；
Step 2：The loose or fine and close callus of selection, with MS as minimal medium, plus the mg/L of 6-BA 0~2.5 and
The mg/L of NAA 0~2.5 are cultivated under the conditions of the lx of illumination 0~2500, induce red cyanine after 7 d on white callus
Element generation；
Step 3：The callus containing anthocyanidin that selecting step 2 is obtained carries out squamous subculture, is basic culture with MS
Base, 1.0 mg/L+NAA of additional 6-BA, 0.5 mg/L+KT 0.3 mg/L+2, the mg/L of 4-D 0~2.0 cultures, flower after 30 d
The substantially accumulation of blue or green element；
Step 4：The callus of pigment accumulation under selection same culture conditions, with hydrochloric acid-methanol as extractant, in difference
Solid-liquid ratio, different extraction time and different Extracting temperatures under optimize pigment extraction conditions, and with ultraviolet specrophotometer 200
Scanned under~650 nm.
Wherein, the condition of culture in step 1 is：24~26 DEG C of temperature, the lx of intensity of illumination 1000, the h/d of light application time 12.
The condition of culture of step 3 is：24~26 DEG C of temperature, the lx of intensity of illumination 2000, the h/d of light application time 12.
The concentration of hydrochloric acid-methanol extractant is 1%~3% in step 4.
Different solid-liquid ratios, different extraction times and different Extracting temperatures refer to solid-liquid ratio 1 in step 4:10~1:50
Between, extraction time between 5~20 h, Extracting temperature is between 4~50 DEG C.
Preferably, the explant of evoked callus is the bud of tuniclike psammosilene root aseptic seedling, callus induction group in step 1
The plant growth regulating substance knitted is the mg/L of 2.5 mg/L+IAA of 6-BA 0.5, and cultivation temperature is 25 DEG C, and intensity of illumination is
2000 lx, light application time is 12 h/d.
Preferably, open-textured callus is chosen in step 2, the Plant growth regulators of anthocyanidin are induced
Matter is the mg/L of 0.5 mg/L+NAA of 6-BA 1.5.
Preferably, the intensity of illumination for inducing anthocyanidin to produce in step 2 is 2000 lx, and light application time is 12 h/
d。
Preferably, the condition of callus accumulation anthocyanidin is the additional 6-BA of MS minimal mediums in step 3
The 1.0 0.5 mg/L and mg/L of KT 0.3 of mg/L, NAA.
Preferably, in step 4 tuniclike psammosilene root callus anthocyanidin maximum absorption band in n=530 nm, pigment content
It is expressed as A530=Abs；With 3% hydrochloric acid-methanol be extractant, solid-liquid ratio be 1:10th, 30 DEG C of the h of extraction time 20, Extracting temperature
When extract.
The beneficial effect that the present invention reaches：
（1）Because the multipair health of synthetic food color has certain harmfulness, and natural pigment has no toxic side effect.
With the bud of tuniclike psammosilene root aseptic seedling as explant, induction produces callus and shoot proliferation, the suitable plant of reselection to the present invention
Growth regulatory substance and illumination condition, so as to induce its callus to accumulate anthocyanidin.
（2）The anthocyanidin that the present invention is obtained is the natural pigment of tuniclike psammosilene root callus cell biosynthesis, is flavonoids
Material, with certain physiologically active, can be used for the industries such as food, medicine, to meet demand of the people to natural pigment.
Brief description of the drawings
Fig. 1 is combined for different plant growth regulating substances（6-BA、NAA）To the shadow of tuniclike psammosilene root callus anthocyanidin induction
Ring；
Fig. 2 is the influence of different illumination intensity and 2,4-D to tuniclike psammosilene root callus anthocyanin accumulation；
Fig. 3 is the ultra-violet absorption spectrum of tuniclike psammosilene root callus anthocyanidin.
Specific embodiment
The present invention is described in further detail with reference to the accompanying drawings and examples.
Technical scheme is comprised the following steps that：
（1）Explant selects the induction with callus：Using tuniclike psammosilene root aseptic seedling（Stem, Ye Heya）As explant,
It is placed in MS culture mediums, adds the mg/L of 6-BA 0~3.0 and mg/L of IAA 0~2.0 in temperature（25 ± 1）DEG C, intensity of illumination
Cultivated under conditions of the 1000 lx and h/d of light application time 12, induction obtains callus；
（2）The induction of anthocyanidin and accumulation：Selection differing texture（Loose or densification）Callus, be basic training with MS
Base, plus the mg/L of 6-BA 0~2.5 and mg/L of NAA 0~2.5 are supported, is cultivated under the lx of illumination 0~2500, white callus after 7 d
Tissue induction red cyanidin generation；Selecting a small amount of callus containing anthocyanidin carries out squamous subculture, is basic with MS
Culture medium, the mg/L+2 of 1.0 mg/L+NAA of additional 6-BA, 0.5 mg/L+KT 0.3, the mg/L of 4-D 0~2.0 are in temperature（25
± 1）DEG C, being cultivated under conditions of the lx of the intensity of illumination 2000 and h/d of light application time 12, anthocyanidin is substantially accumulated after 30 d.
（3）The extraction of tuniclike psammosilene root callus pigment：The callus of pigment accumulation under selection same culture conditions（With filter
Paper blots callus surface moisture）, it is extractant with 1%~3% hydrochloric acid-methanol, in different solid-liquid ratios（1:10~1:50）, it is different
Extraction time（5~20 h）And different Extracting temperatures（4~50 DEG C）Lower optimization pigment extraction conditions, and with uv-spectrophotometric
Meter is scanned under 200~650 nm.
Preferably, the explant of evoked callus is bud, and the Inducing plant growth Auto-regulator of callus is 6-BA
The mg/L of 2.5 mg/L+IAA 0.5, cultivation temperature is（25 ± 1）DEG C, intensity of illumination is 2000 lx, and light application time is 12 h/
d。
It is furthermore preferred that the callus requirement quality chosen is loose, induction anthocyanidin produces plant growth regulating used
Material is the mg/L of 0.5 mg/L+NAA of 6-BA 1.5.As shown in table 1 and Fig. 1.In Fig. 1, A is white callus（6-BA 2.0
mg/L+NAA 1.0 mg/L）；B is aubergine callus（6-BA 0.5 mg/L+NAA 1.0 mg/L）；C is purple callus（6-BA
0.5 mg/L+NAA 1.5 mg/L）
It is furthermore preferred that the illumination condition that induction anthocyanidin is produced is 2000 lx（As shown in table 2 and Fig. 2）.It is furthermore preferred that
The condition of callus anthocyanin accumulation is in the case of without 2,4-D（As shown in table 2 and Fig. 2）, additional 6-BA 1.0
The mg/L of mg/L, NAA 0.5 and mg/L of KT 0.3.
In Fig. 2, D, E, F, G, H, I represent different illumination intensity respectively（500 lx、1000 lx、1500 lx、2000 lx、
2500 lx）To the influence degree of anthocyanin accumulation, when wherein intensity of illumination is 2000 lx（H）Pigment accumulation effect is best；J、K
Influence of 2, the 4-D additions to pigment accumulation is represented, wherein during without 2,4-D（K）Pigment accumulation effect is obvious
In addition, as can be seen from Figure 3, at n=530 nm, pigment content is represented by A to tuniclike psammosilene root pigment maximum absorption band530=
Abs（Absorbance's writes a Chinese character in simplified form, and represents absorbance）；With 3% hydrochloric acid-methanol be extractant, solid-liquid ratio be 1:When the 10th, extracting
Between 20 h, 30 DEG C of Extracting temperature when extraction effect it is preferable.
Embodiment 1：
Vegetable material tuniclike psammosilene root in the present embodiment（Psammosilene tunicoides）Pick up from prestige Ning County of Guizhou Province.
1. explant selection and the induction of callus：
With the aseptic seedling that tuniclike psammosilene root seed is sprouted（Stem, Ye Heya）As explant, because the differentiation and proliferation ability of bud is strong, shape
Into new young young stem and leaf be easy to induction and produce new callus, and propagation is rapid, therefore selection bud is used as evoked callus
Explant material it is preferable.
From the culture dish of a diameter of 9 cm, with MS as minimal medium, 0.5 mg/ of mg/L, IAA of addition 6-BA 2.5
L, the g/L of sucrose 30, the g/L of agar 7, each culture dish are inoculated with 5 buds, and cultivation temperature is（25 ± 1）DEG C, intensity of illumination is
2000 lx, light application time is 12 h/d, produces callus, the follow-up generation Multiplying cultures of 30 d largely to heal to obtain after 7 d of culture
Injured tissue.
2. the induction of anthocyanidin and accumulation：
The induction of anthocyanidin：Because non embryogenic callus quality is loose, cell is relatively large, includes big vacuole, and color
Element is present in vacuole mostly, therefore must select open-textured white callus for the induction of anthocyanidin.Weigh 2.5 g
White callus callus is respectively placed in culture dish, with MS as minimal medium, mg/L, the NAA 1.5 of additional 6-BA 0.5
Mg/L, the g/L of sucrose 30, the g/L of agar 7, cultivation temperature is（25 ± 1）DEG C, intensity of illumination is 2000 lx, and light application time is
12 h/d。
The accumulation of anthocyanidin：Tuniclike psammosilene root white callus needs to carry out squamous subculture to accumulate greatly after producing a small amount of anthocyanidin
The anthocyanidin of amount.Callus of 3.0 g with anthocyanidin is weighed during squamous subculture to be placed in MS minimal mediums, adds 6-BA
1.0 mg/L, NAA0.5 mg/L, KT0.3 mg/L, the g/L of sucrose 30, the g/L of agar 7, cultivation temperature is（25 ± 1）DEG C, light
It is 2000 lx according to intensity, light application time is 12 h/d, and more than 90% Callus formation anthocyanidin can be made after 30~40 d.
3. the extraction of tuniclike psammosilene root callus anthocyanidin：
Selection has the callus of anthocyanin accumulation（Callus surface moisture is blotted with filter paper）, averagely weigh 1 g and heal
Injured tissue is ground in mortar, plus the hydrochloric acid-methanols of 10 mL 3% are transferred in 25 mL test tubes, and is extracted in 30 DEG C of water-baths
20 h, then measure light absorption value at n=530 nm is placed in, each treatment is repeated 3 times.Calculate pigment recovery rate, pigment extraction rate reached
0.41%。
Recovery rate：
In formula：AIt is absorbance of the pigment at 530 nm wavelength；VIt is constant volume；NExtension rate during for colorimetric；
98.2 is extinction coefficient of the pigment at 530 nm wavelength；MIt is callus quality.
Embodiment 2：
The extraction optimization of tuniclike psammosilene root callus anthocyanidin：
There is the callus of anthocyanin accumulation under selection same culture conditions（Callus surface moisture is blotted with filter paper）,
Averagely weigh 1 g callus to be ground in mortar, according to four selected factors（Volume fraction, solid-liquid ratio, time and temperature）
Processed, the results are shown in Table 3.
As known from Table 3, solid-liquid ratio is main affecting factors, and as solid-liquid ratio increases, it is due to solid-liquid two that light absorption value reduces
Mutually there is absorption and dissolution equilibrium, and low temperature contributes to absorption, due to solid-liquid ratio increase, while identical heat is produced, temperature
Degree is reduced, and suction-operated enhancing, absorbance reduces.
The present invention uses plant tissue culture technique production natural plant pigment, and callus proliferation is fast, and pigment is produced
Amount is high, and is not subject to seasonal restrictions, and can in time meet the market demand.
Because natural pigment is bright in colour, have no toxic side effect, and health-care efficacy is protruded, and is gradually pursued by people, plus
Induce with short production cycle, speed fast by tissue culture technique, equipment investment is few, and extraction process is simple, low production cost, produces
Industry DEVELOPMENT PROSPECT is very wide.
The above is only the preferred embodiment of the present invention, it should be pointed out that：For the ordinary skill people of the art
For member, under the premise without departing from the principles of the invention, some improvements and modifications can also be made, these improvements and modifications are also regarded
For the scope that the present invention is protected.
Claims (1)
1. a kind of method that induction tuniclike psammosilene root callus produces anthocyanidin, it is characterised in that：With the nothing that tuniclike psammosilene root seed is sprouted
Vaccine is that material obtains callus through tissue cultures, and anthocyanidin is produced by the regulation evoked callus to condition of culture；
Comprise the following steps that：
Step 1：Using the bud of tuniclike psammosilene root aseptic seedling as explant, it is placed in MS culture mediums, adds the mg/L+IAA 0.5 of 6-BA 2.5
Mg/L is cultivated, and cultivation temperature is 25 DEG C, and intensity of illumination is 2000 lx, and light application time is 12 h/d, and induction obtains callus
Tissue；
Step 2：The loose or fine and close callus of selection, with MS as minimal medium, plus the mg/L+NAA 1.5 of 6-BA 0.5
Mg/L is 2000 lx in intensity of illumination, and light application time is lured after 7 d to be cultivated under the conditions of 12 h/d on white callus
Lead red cyanidin generation；
Step 3：The callus containing anthocyanidin that selecting step 2 is obtained carries out squamous subculture, additional with MS minimal mediums
6-BA 1.0 mg/L, NAA 0.5 the mg/L and mg/L of KT 0.3 cultivated, condition of culture is：24~26 DEG C of temperature, illumination
The lx of intensity 2000, the h/d of light application time 12, anthocyanidin is substantially accumulated after 30 d；
Step 4：The callus of pigment accumulation, tuniclike psammosilene root callus anthocyanidin maximum absorption band under selection same culture conditions
In n=530 nm, pigment content is expressed as A530=Abs；With 3% hydrochloric acid-methanol be extractant, solid-liquid ratio be 1:When the 10th, extracting
Between 20 h, 30 DEG C of Extracting temperature when extract, and scanned under 200~650 nm with ultraviolet specrophotometer.
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| CN106244515A (en) * | 2016-08-04 | 2016-12-21 | 福建省农业科学院农业工程技术研究所 | A kind of method improving Vitis davidi cell culture anthocyanidin content |
| CN110305797B (en) * | 2019-06-18 | 2020-09-08 | 北京理工大学 | Anthocyanin producing strain CJ6 and application thereof |
| CN113913362B (en) * | 2021-10-18 | 2023-11-17 | 大连工业大学 | Sorbus pohuashanensis stem cell for improving anthocyanin content, culture medium and culture method |
| CN113796316A (en) * | 2021-10-18 | 2021-12-17 | 大连工业大学 | Culture medium for promoting psammosilene tunicoides hairy root callus to produce anthocyanin and induction method |
| CN115812601A (en) * | 2022-12-15 | 2023-03-21 | 贵州慧创科技发展有限公司 | Method for inducing generation of anthocyanin by utilizing Psammosilene tunicoides hairy roots |
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| JPH03269060A (en) * | 1990-03-16 | 1991-11-29 | Takeda Chem Ind Ltd | Production of red natural coloring matter |
| JPH07184679A (en) * | 1993-12-27 | 1995-07-25 | Tokyo Gas Co Ltd | Production of anthocyanin-based pigment and medium to be used for the production |
| JPH09308496A (en) * | 1996-05-22 | 1997-12-02 | Ishikawajima Harima Heavy Ind Co Ltd | Production of anthocyanin |
| JP4491655B2 (en) * | 2000-03-31 | 2010-06-30 | 新潟県 | Method for producing anthocyanins using cultured strawberry cells |
| CN104099287B (en) * | 2014-07-07 | 2016-08-24 | 福建省农业科学院农业工程技术研究所 | The acquisition of high yield OPC Vitis davidii Foex callus and subculture keeping method |
| CN104255489B (en) * | 2014-09-16 | 2016-05-18 | 毕节市中药研究所 | The asexual quick breeding technology method of the tender stem of tuniclike psammosilene root band axillalry bud |
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