CN113796316A - Culture medium for promoting psammosilene tunicoides hairy root callus to produce anthocyanin and induction method - Google Patents
Culture medium for promoting psammosilene tunicoides hairy root callus to produce anthocyanin and induction method Download PDFInfo
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Abstract
The invention discloses a culture medium for promoting psammosilene tunicoides hairy root callus to produce anthocyanin, which comprises the following components: the konjak culture medium comprises per liter konjak culture medium: 2.7-8.7g of konjaku flour, 0.10-0.3g of iron powder and 1.5-2.0g of potassium nitrate; an induction method comprising the following processes: inducing the psammosilene tunicoides aseptic seedling to generate a hairy root by using Ar.A4 bacterial liquid and a konjak culture medium, and then proliferating and inducing the hairy root by using the konjak culture medium in sequence to obtain a callus; the callus is placed in a konjak culture medium to induce the generation of anthocyanin and enable the anthocyanin to be accumulated in a large quantity. The natural culture medium is prepared from the natural konjac flour, so that the addition of exogenous substances is reduced, and the cost of the plant tissue culture technology is reduced.
Description
Technical Field
The invention relates to the technical field of tissue culture, in particular to a culture medium and a culture method for promoting psammosilene tunicoides hairy root callus to produce anthocyanin.
Background
The Psammosilene tunicoides is a herbaceous plant of Psammosilene genus of Caryophyllaceae (Caryophylllaceae), is mainly distributed in Guizhou, Yunnan, Sichuan, Tibet and other places in China, and grows in gravel hillsides or calcareous rock seams with the elevation of 2000-3800 m. Psammosilene tunicoides is an endangered medicinal plant, and its root can be used as medicine, and has the functions of dispelling blood stasis, relieving pain, removing toxic material and stopping bleeding. The stem of the wild psammosilene tunicoides plant generally presents purple green, the petals are pink purple during the flowering, and the callus can also generate purple callus under the general illumination condition in the tissue culture process, so the psammosilene tunicoides cell has the capability of synthesizing natural pigment.
The anthocyanin is a natural edible pigment, has certain nutritional and pharmacological effects, and is widely applied to the aspects of food, cosmetics, medicines and the like. At present, most of anthocyanidins used in domestic markets are synthetic pigments, a lot of unknown risks exist, and natural pigments in plant bodies have the advantages of safety, no toxicity and the like. Although anthocyanin exists widely in nature, environmental factors seriously affect the acquisition of raw materials, so that the production of anthocyanin by tissue culture is an effective way. In the prior art, plant tissues are usually induced to generate anthocyanin by MS culture medium, but the addition of macroelements, agar, saccharides and other nutrient elements undoubtedly increases the production cost.
Konjak is a high-quality crop for providing Konjak Glucomannan (KGM). KGM has the important properties of no toxicity, no harm, strong water absorption, rich nutrient substances, promotion of trace element absorption and the like. In the common plant tissue culture medium, macroelements, microelements, iron salt, organic element agar and saccharides are required to be provided to form a complete culture medium, the investment cost of the materials can be saved by utilizing the konjak, and a novel natural konjak culture medium can be formed by improving and utilizing the konjak.
Therefore, the problem to be solved by those skilled in the art is how to provide an induction medium and a culture method using konjac as a main component.
Disclosure of Invention
In view of the above, the invention provides a culture medium for promoting the callus of the Psammosilene tunicoides hairy root to produce anthocyanin, which promotes the dedifferentiation of the hairy root, the dedifferentiation rate reaches 100%, and the induction rate of the callus is improved and reaches 100%; the growth speed is improved by more than 40 percent; the content of the anthocyanin is improved by more than 10 percent, and the cost is saved.
In order to achieve the purpose, the invention adopts the following technical scheme:
a culture medium for promoting the production of anthocyanin from callus of Psammosilene tunicoides hairy roots comprises: the konjak culture medium comprises per liter konjak culture medium: 2.7-8.7g of konjaku flour, 0.10-0.30g of iron powder and 1.5-2.0g of potassium nitrate.
An induction method for promoting the callus of the hairy root of Psammosilene tunicoides to produce anthocyanin comprises the following steps:
1) inducing callus: inducing the psammosilene tunicoides aseptic seedling to generate a hairy root by using Ar.A4 bacterial liquid and a konjak culture medium, and then proliferating and inducing the hairy root by using the konjak culture medium in sequence to obtain a callus;
2) inducing anthocyanin: placing the callus obtained in the step 1) into a konjak culture medium, adding 6-BA1-3mg, IAA0.1-2mg, KT0.1-1mg and 0.01% Ge-132 into each liter of konjak culture medium, and culturing for 15d at 22-25 ℃ under the illumination condition of 2500-;
3) subculturing: transferring the anthocyanin-containing callus into a konjak culture medium, and culturing for 30d at the temperature of 22-25 ℃ and with illumination of 2500-; adding 6-BA1-3mg, IAA0.1-2mg, KT0.1-1mg and 0.01% Ge-132 into per liter of konjak culture medium to accumulate anthocyanin in large amount;
4) and (3) extracting anthocyanin: extracting anthocyanin in the callus obtained in the step 3) by using an extracting agent.
As a preferable technical scheme of the invention, in the step 1), the Ar.A4 bacterial liquid OD600Is 0.6-0.8.
As a preferable technical scheme of the invention, in the step 1), the hairy roots after proliferation are kneaded into a cluster and inoculated into a konjak culture medium for induction, and 2,4-D1-3mg and 6-BA1-2mg are added into each liter of the konjak culture medium.
In a preferred embodiment of the present invention, in step 4), the extractant is a hydrochloric acid-methanol extractant, and the concentration is 1 to 3%.
As a preferable technical scheme of the invention, in the step 4), the material-liquid ratio is 1: 10-1: 15, the extraction time is 5-20h, and the extraction temperature is 4-50 ℃.
According to the technical scheme, the invention has the following technical effects:
(1) the konjak is prepared into a natural culture medium, so that the nutrient substances of the konjak can be utilized to provide macroelements, trace elements and a carbon source for the psammosilene tunicoides aseptic seedlings; in addition, the konjac glucomannan has good coagulability by utilizing the characteristics of the konjac glucomannan, the addition of agar is reduced, the culture cost is saved, and the target product cultured by the konjac glucomannan is safer and more reliable due to a natural culture medium; the konjac culture medium promotes explants to induce callus, and the time is shortened to about 7 d; moreover, the konjak culture medium promotes the growth of callus, and the biomass in unit time is improved by 80%; meanwhile, the konjak culture medium has the effect of inducing anthocyanin synthesis, so that anthocyanin is improved by more than 3 times;
(2) because the artificial synthetic pigments mostly have certain potential safety hazards to human health, the natural pigments basically have no toxic or side effect. The invention takes leaves at the stem ends of the psammosilene tunicoides aseptic seedlings as explants to induce hairy roots, then the hairy roots are used for inducing callus to generate and subculture proliferation, and proper plant hormones and illumination conditions are selected, so that the callus is induced to accumulate anthocyanin, and the generation of side effects is avoided;
(3) ri plasmid in Agrobacterium rhizogenes Ar.A4 has the advantages of rapid proliferation, genetic stability and stimulation of plant cells after infection to synthesize required target hormone, and promotion of growth of cells, so that the addition of Ar.A4 can promote generation of hairy roots, and the hairy roots can proliferate rapidly and synthesize part of plant growth regulator, thereby reducing the use concentration of hormone and obtaining anthocyanin with higher yield in a short period;
drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, it is obvious that the drawings in the following description are only embodiments of the present invention, and for those skilled in the art, other drawings can be obtained according to the provided drawings without creative efforts.
FIG. 1 is a schematic drawing of hairy root induction provided by the present invention;
FIG. 2 is a schematic diagram of a callus provided by the present invention;
FIG. 3 is a schematic diagram of the callus after anthocyanin synthesis provided by the invention.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Example 1 preparation of konjak Medium
(1) Accurately weighing 2.7g of konjac flour, adding 1L of water, and uniformly stirring until the swelling is stable;
(2) heating in a constant-temperature water bath at 55 ℃, adding hydrochloric acid, and stirring for about 2 hours;
(3) adding 0.10mg of iron powder into the culture medium and uniformly mixing;
(4) 1.5g of potassium nitrate was added and the pH was adjusted to 7.5.
Example 2 preparation of konjak Medium
(1) Accurately weighing 8.7g of konjac flour, adding 1L of water, and uniformly stirring until the swelling is stable;
(2) heating in a constant-temperature water bath at 55 ℃, adding hydrochloric acid, and stirring for about 2 hours;
(3) adding 0.30mg of iron powder into the culture medium and uniformly mixing;
(4) 2.0g of potassium nitrate was added and the pH was adjusted to 7.5.
Example 3
Preparation of konjak Medium
(1) Accurately weighing 4.2g of konjac flour, adding 1L of water, and uniformly stirring until the swelling is stable;
(2) heating in a constant-temperature water bath at 55 ℃, adding hydrochloric acid, and stirring for about 2 hours;
(3) adding 0.20mg of iron powder into the culture medium and uniformly mixing;
(4) 1.75g of potassium nitrate was added and the pH was adjusted to 7.5.
Example 4
The method for inducing anthocyanin production by using the konjak culture medium prepared in example 1 comprises the following steps:
1) induction of hairy roots of Psammosilene tunicoides
(11) Respectively pre-culturing young leaves and stems of Psammosilene tunicoides in a konjak culture medium under the condition of illumination for 48 h;
(12) culturing Ar, A4 Agrobacterium rhizogenes bacterial solution at 28 deg.C under 130r/min in dark to OD600＝0.7；
(13) Shaking acetosyringone with concentration of 20mg/L and bacterial liquid for 30 min;
(14) shaking the stem and leaf of the ferro-gold pre-cultured in the step (11) and the bacterial liquid treated in the step (13) for 25 min;
(15) wiping the stems and leaves treated in the step (14) to be dry respectively, and placing the stems and leaves on a konjak culture medium for co-culture until bacterial plaques grow around;
(16) sterilizing the material obtained in the step (15) by using a konjac culture medium added with Cef500mg/L until the agrobacterium is completely removed;
(17) culturing the material in the step (16) until hairy roots are generated, and shearing the hairy roots to carry out resistance screening of high-concentration antibiotics;
(18) carrying out amplification culture on the hairy roots growing vigorously in the step (17), detecting alkali silver nitrate chromogenic opines, and proving that the induction is successful by brown spots; see FIG. 1;
2) explant selection and callus induction
Taking hairy roots induced by Psammosilene tunicoides leaves as explants, selecting a tissue culture bottle, adding 2,4-D2mg/L and 6-BA1.5mg/L into a konjak culture medium, inoculating 5 hairy root tips into each culture medium, culturing at 25 ℃ under a dark condition for 7 days to generate callus, and performing multiplication culture on generations after 30 days to obtain a large amount of callus; see FIG. 2;
3) induction and accumulation of anthocyanidins
Induction of anthocyanins: since non-embryogenic callus is loose in texture, relatively large in cells, containing large vacuoles, and pigments mostly exist in vacuoles, white callus with loose texture must be selected for anthocyanin induction. Weighing 4g of white callus, respectively placing the white callus in a culture medium, adding 6-BA2mg/L, IAA0.6mg/L, KT0.2mg/L and 0.01% Ge-132 into the konjak culture medium, culturing at 23 ℃, with the illumination intensity of 2500Lx and the illumination time of 12 h/d; culturing for 15 d;
4) subculturing: after a small amount of anthocyanin is produced in the Psammosilene tunicoides white callus, subculture is carried out to accumulate a large amount of anthocyanin; weighing 3.0g of callus with anthocyanin and placing the callus into a konjak culture medium during subculture, adding 6-BA2mg/L + IAA0.6mg/L + KT0.2mg/L + 0.01% Ge-132, culturing at 23 ℃, wherein the illumination intensity is 2500Lx, the illumination time is 12h/d, and more than 90% of callus can form anthocyanin after 30d, which is shown in figure 3;
5) extraction of cyanidin from callus of hairy root of Psammosilene tunicoides
Selecting callus with anthocyanin accumulation (sucking out water on the surface of callus with filter paper), weighing 1g of callus on average, grinding in a mortar, adding 10mL of 3% hydrochloric acid-methanol, transferring to a 25mL test tube, extracting in a water bath kettle at 32 ℃ for 18h, measuring absorbance at n-530 nm, and repeating each treatment for 3 times. And (5) calculating the pigment extraction rate, wherein the pigment extraction rate reaches 5.6%.
The calculation formula of the extraction rate is as follows:
note: in the above formula: a is the absorbance of the dye at a wavelength of 530 nm; v is a constant volume; n is the dilution multiple in the colorimetric process; 98.2 is the extinction coefficient of the pigment at the wavelength of 530 nm; m is callus mass.
Example 5
The method for inducing anthocyanin production by using the konjak culture medium prepared in example 1 comprises the following steps:
1) induction of hairy roots of Psammosilene tunicoides
(11) Respectively pre-culturing young leaves and stems of Psammosilene tunicoides in a konjak culture medium under the condition of illumination for 48 h;
(12) culturing Ar, A4 Agrobacterium rhizogenes bacterial solution at 28 deg.C under 130r/min in dark to OD600＝0.6；
(13) Shaking acetosyringone with concentration of 20mg/L and bacterial liquid for 30 min;
(14) shaking the stem and leaf of the ferro-gold pre-cultured in the step (11) and the bacterial liquid treated in the step (13) for 25 min;
(15) wiping the stems and leaves treated in the step (14) to be dry respectively, and placing the stems and leaves on a konjak culture medium for co-culture until bacterial plaques grow around;
(16) sterilizing the material obtained in the step (15) by using a konjac culture medium added with Cef500mg/L until the agrobacterium is completely removed;
(17) culturing the material in the step (16) until hairy roots are generated, and shearing the hairy roots to carry out resistance screening of high-concentration antibiotics;
(18) carrying out amplification culture on the hairy roots growing vigorously in the step (17), detecting alkali silver nitrate chromogenic opines, and proving that the induction is successful by brown spots;
2) explant selection and callus induction
Taking hairy roots induced by Psammosilene tunicoides leaves as explants, selecting a tissue culture bottle, adding 2, 4-D3 mg/L and 6-BA2mg/L into a konjak culture medium, inoculating 5 hairy root tips into each culture medium, culturing at 22 ℃ under a dark condition for 7 days to generate callus, and performing generational propagation culture after 30 days to obtain a large amount of callus;
3) induction and accumulation of anthocyanidins
Induction of anthocyanins: since non-embryogenic callus is loose in texture, relatively large in cells, containing large vacuoles, and pigments mostly exist in vacuoles, white callus with loose texture must be selected for anthocyanin induction. Weighing 4g of white callus, respectively placing the white callus in a culture medium, adding 6-BA3mg/L, IAA2mg/L, KT1mg/L and 0.01% Ge-132 into the konjac culture medium, culturing at 25 ℃, under the illumination intensity of 2800Lx and for 12h/d, and culturing for 15 d;
4) subculturing: after the Psammosilene tunicoides white callus produces a small amount of anthocyanin, subculture is carried out to accumulate a large amount of anthocyanin. In the process of subculture, 3.0g of callus with anthocyanin is weighed and placed in a konjak culture medium, 6-BA3mg/L + IAA2mg/L + KT1mg/L + 0.01% Ge-132 is added, the culture temperature is 25 ℃, the illumination intensity is 2800Lx, the illumination time is 12h/d, and more than 90% of callus can form anthocyanin after 30 d.
5) Extraction of cyanidin from callus of hairy root of Psammosilene tunicoides
Selecting callus with anthocyanin accumulation (sucking out water on the surface of callus with filter paper), weighing 1g of callus on average, grinding in a mortar, adding 25mL of 1% hydrochloric acid-methanol, transferring to a 25mL test tube, extracting in a 50 ℃ water bath for 5h, measuring absorbance at n-530 nm, and repeating each treatment for 3 times. And calculating the pigment extraction rate, wherein the pigment extraction rate is 5.6%.
The calculation formula of the extraction rate is as follows:
note: in the above formula: a is the absorbance of the dye at a wavelength of 530 nm; v is a constant volume; n is the dilution multiple in the colorimetric process; 98.2 is the extinction coefficient of the pigment at the wavelength of 530 nm; m is callus mass.
Example 6
The method for inducing anthocyanin production by using the konjak culture medium prepared in example 1 comprises the following steps:
1) induction of hairy roots of Psammosilene tunicoides
(11) Respectively pre-culturing young leaves and stems of Psammosilene tunicoides in a konjak culture medium under the condition of illumination for 48 h;
(12) culturing Ar, A4 Agrobacterium rhizogenes bacterial solution at 28 deg.C under 130r/min in dark to OD600＝0.8；
(13) Shaking acetosyringone with concentration of 20mg/L and bacterial liquid for 30 min;
(14) shaking the stem and leaf of the ferro-gold pre-cultured in the step (11) and the bacterial liquid treated in the step (13) for 25 min;
(15) wiping the stems and leaves treated in the step (14) to be dry respectively, and placing the stems and leaves on a konjak culture medium for co-culture until bacterial plaques grow around;
(16) sterilizing the material obtained in the step (15) by using a konjac culture medium added with Cef500mg/L until the agrobacterium is completely removed;
(17) culturing the material in the step (16) until hairy roots are generated, and shearing the hairy roots to carry out resistance screening of high-concentration antibiotics;
(18) carrying out amplification culture on the hairy roots growing vigorously in the step (17), detecting alkali silver nitrate chromogenic opines, and proving that the induction is successful by brown spots;
2) explant selection and callus induction
Taking hairy roots induced by Psammosilene tunicoides leaves as explants, selecting a tissue culture bottle, adding 2,4-D1 mg/L and 6-BA1mg/L to a konjak culture medium, inoculating 5 hairy root tips to each culture medium, culturing at 23 ℃ under a dark condition to generate callus after 7 days, and performing generational propagation culture after 30 days to obtain a large amount of callus;
3) induction and accumulation of anthocyanidins
Induction of anthocyanins: since non-embryogenic callus is loose in texture, relatively large in cells, containing large vacuoles, and pigments mostly exist in vacuoles, white callus with loose texture must be selected for anthocyanin induction. Weighing 4g of white callus, respectively placing the white callus in a culture medium, adding 6-BA1mg/L, IAA0.1mg/L, KT0.1mg/L and 0.01% Ge-132 into the konjak culture medium, culturing at 22 ℃, with the illumination intensity of 3000Lx and the illumination time of 12h/d for 15 d;
4) subculturing: after the Psammosilene tunicoides white callus produces a small amount of anthocyanin, subculture is carried out to accumulate a large amount of anthocyanin. In the process of subculture, 3.0g of callus with anthocyanin is weighed and placed in a konjak culture medium, 6-BA3mg/L + IAA2mg/L + KT1mg/L + 0.01% Ge-132 is added, the culture temperature is 25 ℃, the illumination intensity is 2800Lx, the illumination time is 12h/d, and more than 90% of callus can form anthocyanin after 30 d.
5) Extraction of cyanidin from callus of hairy root of Psammosilene tunicoides
Selecting callus with anthocyanin accumulation (sucking out water on the surface of callus with filter paper), weighing 1g of callus on average, grinding in a mortar, adding 25mL of 1% hydrochloric acid-methanol, transferring to a 25mL test tube, extracting in a 50 ℃ water bath for 5h, measuring absorbance at n-530 nm, and repeating each treatment for 3 times. And (4) calculating the pigment extraction rate, wherein the pigment extraction rate is 46-51%.
Example 7
The method for inducing anthocyanin production by using the konjak culture medium prepared in example 1 comprises the following steps:
1) induction of hairy roots of Psammosilene tunicoides
(11) Respectively pre-culturing young leaves and stems of Psammosilene tunicoides in a konjak culture medium under the condition of illumination for 48 h;
(12) culturing Ar, A4 Agrobacterium rhizogenes bacterial solution at 28 deg.C under 130r/min in dark to OD600＝0.8；
(13) Shaking acetosyringone with concentration of 20mg/L and bacterial liquid for 30 min;
(14) shaking the stem and leaf of the ferro-gold pre-cultured in the step (11) and the bacterial liquid treated in the step (13) for 25 min;
(15) wiping the stems and leaves treated in the step (14) to be dry respectively, and placing the stems and leaves on a konjak culture medium for co-culture until bacterial plaques grow around;
(16) sterilizing the material obtained in the step (15) by using a konjac culture medium added with Cef500mg/L until the agrobacterium is completely removed;
(17) culturing the material in the step (16) until hairy roots are generated, and shearing the hairy roots to carry out resistance screening of high-concentration antibiotics;
(18) carrying out amplification culture on the hairy roots growing vigorously in the step (17), detecting alkali silver nitrate chromogenic opines, and proving that the induction is successful by brown spots;
2) explant selection and callus induction
Taking hairy roots induced by Psammosilene tunicoides leaves as explants, selecting a tissue culture bottle, adding 2, 4-D3 mg/L and 6-BA2mg/L into a konjak culture medium, inoculating 5 hairy root tips into each culture medium, culturing at the temperature of (25 +/-1) DEG C under a dark condition for 7 days to generate callus, and performing multiplication culture on successive generations after 30 days to obtain a large amount of callus;
3) induction and accumulation of anthocyanidins
Induction of anthocyanins: since non-embryogenic callus is loose in texture, relatively large in cells, containing large vacuoles, and pigments mostly exist in vacuoles, white callus with loose texture must be selected for anthocyanin induction. Weighing 4g of white callus, respectively placing the white callus in a culture medium, adding 6-BA2mg/L, IAA0.6mg/L, KT0.2mg/L and 0.01% Ge-132 into the konjak culture medium, wherein the culture temperature is (23 +/-1) DEG C, the illumination intensity is 2500Lx, and the illumination time is 12 h/d.
4) Accumulation of anthocyanins: after the Psammosilene tunicoides white callus produces a small amount of anthocyanin, subculture is carried out to accumulate a large amount of anthocyanin. In the process of subculture, 3.0g of callus with anthocyanin is weighed and placed in a konjak culture medium, 6-BA2mg/L + IAA0.6mg/L + KT0.2mg/L + 0.01% Ge-132 is added, the culture temperature is (23 +/-1) DEG C, the illumination intensity is 2500Lx, the illumination time is 12h/d, and more than 90% of callus can form anthocyanin after 30-40 d.
5) Extraction of cyanidin from callus of hairy root of Psammosilene tunicoides
Selecting callus with anthocyanin accumulation (sucking out water on the surface of callus with filter paper), weighing 1g of callus on average, grinding in a mortar, adding 50mL of 3% hydrochloric acid-methanol, transferring to a 25mL test tube, extracting in a water bath kettle at 4 ℃ for 20h, measuring absorbance at n-530 nm, and repeating each treatment for 3 times. And calculating the pigment extraction rate, wherein the pigment extraction rate is 5.6%.
The calculation formula of the extraction rate is as follows:
note: in the above formula: a is the absorbance of the dye at a wavelength of 530 nm; v is a constant volume; n is the dilution multiple in the colorimetric process; 98.2 is the extinction coefficient of the pigment at the wavelength of 530 nm; m is callus mass.
Example 7
The culture was performed in the same manner as in example 4 to obtain test group 1;
the culture was performed by the method of example 4, but directly with explants, callus was induced without inducing generation of hairy roots, test group 2;
the culture was performed in the same manner as in example 4, but the explant was directly used for the culture, callus was induced without inducing the production of hairy roots, and the konjak medium was changed to the MS medium to obtain test group 3;
the culture was performed by the method of example 4, and the konjak medium was changed to the MS medium to obtain test group 4;
the biomass growth, anthocyanin content and callus generation time in each group are counted, and the results are shown in table 1;
TABLE 1
As can be seen from Table 1, the natural culture medium prepared from the natural konjac flour reduces the addition of exogenous substances, reduces the cost of plant tissue culture technology, and provides safer and more reliable basis for producing natural substances; the natural pigment is produced by utilizing a tissue culture technology, the characteristic of hairy roots and the characteristic of rapid callus proliferation are utilized, the yield of the target product is higher, the influence of the external environment is avoided, and the market demand can be met.
The embodiments in the present description are described in a progressive manner, each embodiment focuses on differences from other embodiments, and the same and similar parts among the embodiments are referred to each other.
The previous description of the disclosed embodiments is provided to enable any person skilled in the art to make or use the present invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the generic principles defined herein may be applied to other embodiments without departing from the spirit or scope of the invention. Thus, the present invention is not intended to be limited to the embodiments shown herein but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.
Claims (7)
1. A culture medium for promoting generation of anthocyanin from Psammosilene tunicoides hairy root callus is characterized by comprising the following components: konjak culture medium, wherein, per liter konjak culture medium includes: 2.7-8.7g of konjaku flour, 0.10-0.30g of iron powder and 1.5-2.0g of potassium nitrate.
2. An induction method for promoting the callus of the hairy root of Psammosilene tunicoides to produce anthocyanin is characterized by comprising the following steps:
1) inducing callus: adding 0.01-0.05% acetosyringone to incubate Ar.A4 bacterial liquid for 30min and psammosilene tunicoides aseptic seedlings on a konjak culture medium for 22-25 ℃, co-culturing for 12-14h under a dark condition, then using 0.6-0.8% cefotaxime sodium for sterilization, and transferring to the konjak culture medium to induce generation of hairy roots; cutting off the induced hairy roots, transferring the cut hairy roots to a konjak culture medium for enrichment culture, and then transferring the cut hairy roots to the konjak culture medium for inducing callus;
2) inducing anthocyanin: placing the callus obtained in the step 1) into a konjak culture medium, adding 1-3mg of 6-BA, 0.1-2mg of IAA, 0.1-1mg of KT and 0.01% Ge-132 into each liter of konjak culture medium, and culturing for 15d at 22-25 ℃ under the illumination condition of 2500-;
3) subculturing: transferring the callus containing anthocyanin into a konjak culture medium, culturing for 30d at the temperature of 22-25 ℃ and under illumination of 2500-;
4) and (3) extracting anthocyanin: extracting anthocyanin in the callus obtained in the step 3) by using an extracting agent.
3. The induction method for promoting the production of anthocyanin from Psammosilene tunicoides hairy root callus according to claim 2, wherein in step 1), the bacterial liquid OD of Ar.A4 is used600Is 0.6-0.8.
4. The method for inducing the callus of Psammosilene tunicoides hairy root to produce anthocyanin as claimed in claim 2, wherein in step 1), the hairy root after proliferation is kneaded into clusters and inoculated into konjak culture medium for inducing, 2,4-D1-3mg and 6-BA1-2mg are added into each liter of konjak culture medium.
5. The induction method for promoting the anthocyanin production from the Psammosilene tunicoides hairy root callus tissue as claimed in claim 2, wherein in the step 4), the extractant is hydrochloric acid-methanol extractant and the concentration is 1-3%.
6. The induction method for promoting the generation of the anthocyanin from the Psammosilene tunicoides hairy root callus tissue as claimed in claim 2, wherein in the step 4), the material-liquid ratio is 1: 10-1: 50, the extraction time is 5-20h, and the extraction temperature is 4-50 ℃.
7. A culture medium for promoting anthocyanin accumulation in callus of hairy roots of Psammosilene tunicoides is characterized in that on the basis of a konjak culture medium, 1-3mg of 6-BA, 0.1-2mg of IAA, 0.1-1mg of KT and 0.01% of Ge-132 are added into each liter of konjak culture medium.
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