CN116649217A - Proliferation culture method of fig callus - Google Patents
Proliferation culture method of fig callus Download PDFInfo
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- 230000035755 proliferation Effects 0.000 title claims abstract description 102
- 238000012136 culture method Methods 0.000 title claims abstract description 18
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- 238000012258 culturing Methods 0.000 claims abstract description 13
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- 229910052782 aluminium Inorganic materials 0.000 description 2
- HUMNYLRZRPPJDN-UHFFFAOYSA-N benzaldehyde Chemical compound O=CC1=CC=CC=C1 HUMNYLRZRPPJDN-UHFFFAOYSA-N 0.000 description 2
- 239000003153 chemical reaction reagent Substances 0.000 description 2
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- UQHKFADEQIVWID-UHFFFAOYSA-N cytokinin Natural products C1=NC=2C(NCC=C(CO)C)=NC=NC=2N1C1CC(O)C(CO)O1 UQHKFADEQIVWID-UHFFFAOYSA-N 0.000 description 2
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- IIDAJRNSZSFFCB-UHFFFAOYSA-N 4-amino-5-methoxy-2-methylbenzenesulfonamide Chemical compound COC1=CC(S(N)(=O)=O)=C(C)C=C1N IIDAJRNSZSFFCB-UHFFFAOYSA-N 0.000 description 1
- DBMJZOMNXBSRED-UHFFFAOYSA-N Bergamottin Natural products O1C(=O)C=CC2=C1C=C1OC=CC1=C2OCC=C(C)CCC=C(C)C DBMJZOMNXBSRED-UHFFFAOYSA-N 0.000 description 1
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Classifications
-
- 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
- A01H4/002—Culture media for tissue culture
-
- 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
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
- Y02P60/00—Technologies relating to agriculture, livestock or agroalimentary industries
- Y02P60/40—Afforestation or reforestation
Abstract
The invention discloses a proliferation culture method of fig callus, which relates to the technical field of plant biology, and is technically characterized by comprising the following steps: s1, callus induction: inoculating the explant on an induction culture medium, and carrying out dark culture for 20-30 days at the temperature of 23-25 ℃ to obtain callus; s2, callus proliferation: spreading the callus obtained by culturing in the step S1 on a proliferation culture medium, and culturing for 25-35 days; the formula of the proliferation culture medium consists of a basic culture medium and plant growth hormone, wherein the basic culture medium comprises the following components: MS culture medium powder, sucrose, agar, and one or more of the following components of plant growth hormone: 1-naphthylacetic acid, zeatin, 6-benzylaminopurine, thidiazuron. The fig callus cultivated by the cultivation method provided by the invention has high content of active substances.
Description
Technical Field
The invention relates to the technical field of plant biology, in particular to a proliferation culture method of fig callus.
Background
Plants are the largest source of natural in the human social food, pharmaceutical and cosmetic fields. The use of plants and plant extracts in traditional medicine has been known since ancient times. Fig is native to the Mediterranean coast and is one of the oldest fruit trees in the world. The roots, stems, leaves and fruits of fig can be used as medicines, and contain various medicinal components such as psoralen, bergapten, benzaldehyde, furocoumarin and the like. The fig extract has an inhibitory effect on cancer cells, and is therefore also considered as an anticancer food. In addition, the SOD (superoxide dismutase) in fig has higher activity and good effects of resisting aging, eliminating freckles and the like, and is becoming an important raw material of high-grade cosmetics.
Upon traumatic stimulation of plant cells, thin-walled cells, known as callus, are formed on the wound surface, which are increased in volume and can originate from living cells of various tissues within any organ of the plant body. Although in a suitable medium, the calli may be maintained indefinitely in vitro. However, in practical operation, it is not easy to fudge the culture conditions required for mass propagation of calli. In the prior art, the fig callus is not efficiently propagated, and the volume of one propagation period is only increased by 1-2 times.
In view of this, the present invention has been made.
Disclosure of Invention
The invention aims to solve the problems, and provides a proliferation culture method for fig callus, which solves the problems of low proliferation efficiency and low content of active ingredients of fig callus in the prior art.
In order to achieve the above object, the present invention is as follows: a proliferation culture method of fig callus comprises the following steps: s1, callus induction: inoculating the explant on an induction culture medium, and carrying out dark culture for 20-30 days at the temperature of 23-25 ℃ to obtain callus; s2, callus proliferation: spreading the callus obtained by culturing in the step S1 on a proliferation culture medium, and culturing for 25-35 days; wherein the proliferation culture medium consists of a basic culture medium and a plant growth regulator; the basic culture medium comprises 4.41g/L of MS culture medium powder, 25-30g/L of sucrose and 6-8g/L of agar; the plant growth regulator comprises one or more of the following components: 1-naphthylacetic acid 0.05-0.1mg/L, zeatin 3.7-4.3mg/L, 6-benzylaminopurine 3.7-8.3mg/L and thidiazuron 0.7-4.3mg/L.
Preferably, the proliferation medium consists of a minimal medium and a plant growth regulator; the basic culture medium comprises 4.41g/L of MS culture medium powder, 25-30g/L of sucrose and 6-8g/L of agar, and the plant growth regulator comprises 0.1mg/L of 1-naphthylacetic acid and 3.7-4.3mg/L of zeatin.
Preferably, the proliferation medium consists of a minimal medium and a plant growth regulator; the basic culture medium comprises 4.41g/L of MS culture medium powder, 25-30g/L of sucrose and 6-8g/L of agar, and the plant growth regulator comprises 3.7-4.3mg/L of 6-benzylaminopurine and 0.1mg/L of 1-naphthylacetic acid.
Preferably, the proliferation medium consists of a minimal medium and a plant growth regulator; the basic culture medium comprises 4.41g/L of MS culture medium powder, 25-30g/L of sucrose and 6-8g/L of agar, and the plant growth regulator comprises 5.7-6.3mg/L of 6-benzylaminopurine and 0.1mg/L of 1-naphthylacetic acid.
Preferably, the proliferation medium consists of a minimal medium and a plant growth regulator; the basic culture medium comprises 4.41g/L of MS culture medium powder, 25-30g/L of sucrose and 6-8g/L of agar, and the plant growth regulator comprises 7.7-8.3mg/L of 6-benzylaminopurine and 0.1mg/L of 1-naphthylacetic acid.
Preferably, the proliferation medium consists of a minimal medium and a plant growth regulator; the basic culture medium comprises 4.41g/L of MS culture medium powder, 25-30g/L of sucrose and 6-8g/L of agar, and the plant growth regulator comprises 0.05mg/L of 1-naphthylacetic acid, 3.7-4.7mg/L of 6-benzylaminopurine and 0.7-1.3mg/L of thidiazuron.
Preferably, the proliferation medium consists of a minimal medium and a plant growth regulator; the basic culture medium comprises 4.41g/L of MS culture medium powder, 25-30g/L of sucrose and 6-8g/L of agar, and the plant growth regulator comprises 0.05mg/L of 1-naphthylacetic acid, 3.7-4.3mg/L of 6-benzylaminopurine and 3.7-4.3mg/L of thidiazuron.
Preferably, the induction medium is formulated as follows: MS culture medium powder 4.41g/L, sucrose 25-30g/L, agar 6-8g/L, 6-benzyl aminopurine 1.5-2.5mg/L, 1-naphthylacetic acid 0.45-0.55mg/L.
Preferably, the induction medium is formulated by the steps of: after the MS culture medium powder, sucrose and agar are prepared, the pH is adjusted to 5.8-6, and 6-benzyl amino purine and 1-naphthylacetic acid are added when the temperature is reduced to 55-65 ℃ for sterilization.
Preferably, the explant is a stem segment or leaf of fig, and the length of the explant is 8-12mm.
Compared with the prior art, the invention has the beneficial effects that:
1. in the proliferation culture method, a plurality of cytokinins are selected for proliferation culture of the callus, and the ingredients are synergistic, so that compared with the formula of a plurality of fig proliferation culture mediums, the fresh weight of the callus obtained in one proliferation period can be increased by 5.52 times at most, and the content of active ingredients in the callus is obviously increased.
2. By the callus culture proliferation method, seasons or plant reproductive cycles can be omitted, and fresh plant callus can be obtained at any time. In addition, the growth conditions of plant callus are easily standardized, without risk of pathogenic or environmental pollution, and can be extended to industrial scale.
Drawings
FIG. 1 is a schematic diagram of the callus of Ficus carica obtained after proliferation in test examples 1 to 16 according to the present invention.
Detailed Description
In order that those skilled in the art will better understand the present invention, a technical invention will be described in further detail below with reference to the following embodiments of the present invention and the accompanying drawings, wherein it is apparent that the described embodiments are only some embodiments of the present invention, and not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the present invention without making any inventive effort, shall fall within the scope of the present invention.
It should be noted that, without conflict, the embodiments of the present invention and features of the embodiments may be combined with each other. The present invention will be described in detail with reference to examples.
The terminology involved in the embodiments of the invention is further described as follows:
6-BA: 6-benzylaminopurine;
NAA: 1-naphthalene acetic acid;
TDZ: thidiazuron;
ZT: zeatin.
The culture medium selected in the embodiment of the invention is as follows:
induction medium: consists of a basic culture medium and a plant growth regulator, wherein the basic culture medium comprises: MS culture medium powder, sucrose, agar, and plant growth regulator including 6-BA and NAA.
Proliferation medium: consists of a basic culture medium and a plant growth regulator, wherein the basic culture medium comprises: MS culture medium powder, sucrose, agar, and plant growth regulator including 6-BA, NAA, TDZ, ZT.
By using the culture medium, plant callus, preferably fig callus is subjected to proliferation culture, and the following examples are specifically shown below:
example 1
The proliferation method of the fig callus comprises the following steps:
s1, callus induction: inoculating 4g of the explant on an induction medium for dark culture at 25 ℃ for 25 days; the induction culture medium consists of a basic culture medium and a plant growth regulator, wherein the basic culture medium is prepared from the following components: MS culture medium powder 4.41g/L, sucrose 270g/L, agar 8g/L, and plant growth regulator consisting of 2 mg/L6-BA and 0.5mg/L NAA. The induction culture medium is prepared by the following steps: after the preparation of the basic culture medium is completed, the pH value of the basic culture medium is adjusted to 5.8, and the basic culture medium is added with a plant growth regulator and sterilized for 20min at 120 ℃ and 0.1 Mpa.
S2, callus proliferation: the callus obtained by culturing in the step S1 is paved on a proliferation culture medium for culturing for 25 days at the temperature of 25 ℃, and the formula of the proliferation culture medium is as follows: MS culture medium powder 4.41g/L, sucrose 28g/L, agar 7g/L, 6-BA4mg/L, TDZ mg/L, NAA 0.05.05 mg/L. The proliferation medium is prepared by the following steps: after the preparation of the basic culture medium is completed, the pH value of the basic culture medium is adjusted to 5.8, NAA, TDZ and 6-BA are added, and the basic culture medium is sterilized for 20min at 120 ℃ and 0.1 Mpa.
Callus proliferation coefficient = fresh weight of callus 25 days after inoculation/fresh weight of callus before inoculation. The callus proliferation factor in example 1 was 5.52, total phenol content was 92.26.+ -. 0.42mg/g, total flavone content was 428.81.+ -. 1.44mg/g.
Example 2
The proliferation method of the fig callus comprises the following steps:
s1, callus induction: inoculating 4g of the explant on an induction medium followed by dark culture at 25℃for 25 days; the induction culture medium consists of a basic culture medium and a plant growth regulator, wherein the basic culture medium is prepared from the following components: MS culture medium powder 4.41g/L, sucrose 25-30g/L, agar 6-8g/L, and plant growth regulator composed of 2 mg/L6-BA and 0.5mg/L NAA. The induction culture medium is prepared by the following steps: after the preparation of the basic culture medium is completed, the pH value of the basic culture medium is adjusted to 5.8-6, and then the basic culture medium is added with a plant growth regulator and sterilized for 20min at 120 ℃ and 0.1 Mpa.
S2, callus proliferation: spreading the callus obtained by culturing in the step S1 on a proliferation culture medium, and then carrying out dark culture for 25 days at 25 ℃; the proliferation medium formulation is as follows: MS culture medium powder 4.41g/L, sucrose 28g/L, agar 8g/L, 6-BA4mg/L and NAA0.1mg/L. The proliferation medium is prepared by the following steps: after the preparation of the basic culture medium is completed, the pH value of the basic culture medium is adjusted to 5.8, NAA and 6-BA are added, and the basic culture medium is sterilized for 20min at 120 ℃ and 0.1 Mpa.
Callus proliferation coefficient = fresh weight of callus 25 days after inoculation/fresh weight of callus before inoculation. The callus proliferation factor in example 2 was 3.45, the total phenol amount was 98.22mg/g, and the total flavone amount was 393.13mg/g.
Example 3
The proliferation method of the fig callus comprises the following steps:
s1, callus induction: inoculating 4g of the explant on an induction medium and then performing dark culture for 25 days at the temperature of 23-25 ℃; the induction culture medium consists of a basic culture medium and a plant growth regulator, wherein the basic culture medium is prepared from the following components: MS culture medium powder 4.41g/L, sucrose 25g/L, agar 6g/L, and plant growth regulator consisting of 2 mg/L6-BA and 0.5mg/L NAA. The induction culture medium is prepared by the following steps: after the preparation of the basic culture medium is completed, the pH value of the basic culture medium is adjusted to 5.8-6, and then the basic culture medium is added with a plant growth regulator and sterilized for 20min at 120 ℃ and 0.1 Mpa.
S2, callus proliferation: the callus obtained by culturing in S1 is spread on a proliferation medium, and then dark culture is carried out for 25 days at the temperature of 23 ℃, wherein the proliferation medium comprises the following formula: MS culture medium powder 4.41g/L, sucrose 28g/L, agar 8g/L, 6-BA6mg/L and NAA0.1mg/L. The proliferation medium is prepared by the following steps: after the preparation of the basic culture medium is completed, regulating the pH value to 5.8-6, adding NAA and 6-BA, and sterilizing at 120 ℃ and 0.1Mpa for 20 min.
Callus proliferation coefficient = fresh weight of callus 25 days after inoculation/fresh weight of callus before inoculation. The callus proliferation factor in example 3 was 3.17, the total phenol amount was 101.26mg/g, and the total flavone amount was 439.41mg/g.
Example 4
The proliferation method of the fig callus comprises the following steps:
s1, callus induction: inoculating 4g of the explant on an induction medium followed by dark culture at 25℃for 25 days; the induction culture medium consists of a basic culture medium and a plant growth regulator, wherein the basic culture medium is prepared from the following components: MS culture medium powder 4.41g/L, sucrose 30g/L, agar 8g/L, and plant growth regulator consisting of 2 mg/L6-BA and 0.5mg/L NAA. The induction culture medium is prepared by the following steps: after the preparation of the basic culture medium is completed, the pH value of the basic culture medium is adjusted to 5.8-6, and then the basic culture medium is added with a plant growth regulator and sterilized for 20min at 120 ℃ and 0.1 Mpa.
S2, callus proliferation: spreading the callus obtained by culturing in the step S1 on a proliferation culture medium, and then carrying out dark culture for 25 days at 25 ℃; the proliferation medium formulation is as follows: MS culture medium powder 4.41g/L, sucrose 28g/L, agar 6-8g/L, 6-BA 8mg/L, NAA and 0.1mg/L. The proliferation medium is prepared by the following steps: after the preparation of the basic culture medium is completed, regulating the pH value to 5.8-6, adding NAA and 6-BA, and sterilizing at 120 ℃ and 0.1Mpa for 20 min.
Callus proliferation coefficient = fresh weight of callus 25 days after inoculation/fresh weight of callus before inoculation. The callus proliferation factor in example 4 was 4.03, the total phenol amount was 111.01mg/g, and the total flavone amount was 505.12mg/g.
Example 5
The proliferation method of the fig callus comprises the following steps:
s1, callus induction: inoculating 4g of the explant on an induction medium followed by dark culture at 25℃for 25 days; the induction culture medium consists of a basic culture medium and a plant growth regulator, wherein the basic culture medium is prepared from the following components: MS culture medium powder 4.41g/L, sucrose 25-30g/L, agar 6-8g/L, and plant growth regulator composed of 2 mg/L6-BA and 0.5mg/L NAA. The induction culture medium is prepared by the following steps: after the preparation of the basic culture medium is completed, the pH value of the basic culture medium is adjusted to 5.8-6, and then the basic culture medium is added with a plant growth regulator and sterilized for 20min at 120 ℃ and 0.1 Mpa.
S2, callus proliferation: spreading the callus obtained by culturing in the step S1 on a proliferation culture medium, and then carrying out dark culture for 25 days at 25 ℃; the proliferation medium formulation is as follows: MS culture medium powder 4.41g/L, sucrose 28g/L, agar 7g/L, TDZ mg/L, 6-BA4mg/L and NAA0.05mg/L. The proliferation medium is prepared by the following steps: after the preparation of the basic culture medium is completed, regulating the pH value to 5.8-6, adding NAA, TDZ and 6-BA, and sterilizing at 120deg.C and 0.1Mpa for 20 min.
Callus proliferation coefficient = fresh weight of callus 25 days after inoculation/fresh weight of callus before inoculation. The callus proliferation factor in example 4 was 3.55, the total phenol amount was 91.87mg/g, and the total flavone amount was 398.25mg/g.
Example 6
The proliferation method of the fig callus comprises the following steps:
s1, callus induction: inoculating 4g of the explant on an induction medium followed by dark culture at 25℃for 25 days; the induction culture medium consists of a basic culture medium and a plant growth regulator, wherein the basic culture medium is prepared from the following components: MS culture medium powder 4.41g/L, sucrose 25-30g/L, agar 6g/L, and plant growth regulator consisting of 2 mg/L6-BA and 0.5mg/L NAA. The induction culture medium is prepared by the following steps: after the preparation of the basic culture medium is completed, the pH value of the basic culture medium is adjusted to 5.8-6, and then the basic culture medium is added with a plant growth regulator and sterilized for 20min at 120 ℃ and 0.1 Mpa.
S2, callus proliferation: spreading the callus obtained by culturing in the step S1 on a proliferation culture medium, and then carrying out dark culture for 25 days at 25 ℃; the proliferation medium formulation is as follows: MS culture medium powder 4.41g/L, sucrose 28g/L, agar 6-8g/L, ZT mg/L, NAA 0.1.1. The proliferation medium is prepared by the following steps: after the preparation of the basic culture medium is completed, regulating the pH value to 5.8-6, adding NAA, sterilizing at 120 ℃ and 0.1Mpa for 20min, and finally adding ZT.
Callus proliferation coefficient = fresh weight of callus 25 days after inoculation/fresh weight of callus before inoculation. The callus proliferation factor in example 4 was 5.32, the total phenol amount was 109.29mg/g, and the total flavone amount was 455.40mg/g.
Comparative examples
Comparative example the same test was performed as the example, except that: the comparative example adjusts the type and amount of growth regulator in the proliferation medium. The types and amounts of the growth regulators added to the proliferation medium in the comparative examples and their proliferation coefficients are shown in Table 1, and calli obtained after proliferation of each test example are shown in FIG. 1.
TABLE 1 influence of different hormone concentration ratios on Ficus carica callus proliferation factor
From Table 1, it can be seen that the callus proliferation factor of TDZ,6-BA and NAA combinations is significantly higher than that of other combinations, and the hormone ratio of the callus proliferation medium with the optimal proliferation factor is: TDZ-4mg/L+6-BA-4mg/L+NAA-0.05mg/L. The proliferation factor was 5.52 and the calli appeared yellowish brown.
Experimental example 1 determination of weight gain of callus
Fresh weight measurement: on days 5, 15 and 25 of callus culture, the fig callus cultured by different culture medium formulations are randomly taken out, at least 3 bottles of each culture medium are taken out, and the weight of the callus is measured, and the specific table 2 is shown.
TABLE 2 Effect of different concentration hormone ratios on Ficus carica callus weight gain
As shown in table 2, when the fig callus is transferred to the proliferation medium, the proliferation coefficient of the callus is smaller in the first 5 days, the callus is in a slow growth stage, the growth amount of the callus in the 5 th to 15 th days of the proliferation stage is obviously lower than that in the 15 th to 25 th days, the fresh weight of the callus can reach 10.11g at the maximum at 15 th days, and the hormone ratio of the callus proliferation medium is as follows: ZT-4mg/L+NAA-0.1mg/L; the maximum fresh weight of the callus can reach 22.09g at the 25 th day, and the hormone proportion of the callus proliferation culture medium is TDZ-4mg/L+6-BA-4mg/L+NAA-0.05mg/L.
Experimental example 2 determination of active ingredient
And (3) freeze drying: taking fresh fig callus, pre-freezing for 24 hours in a constant temperature refrigerator at the temperature of minus 30 ℃, then drying for 48 hours in a vacuum freeze dryer, wherein the cold trap temperature is minus 60 ℃, the freezing warehouse temperature is minus 45 ℃ to minus 35 ℃, and the vacuum degree is 0 to 10pa.
Extracting: accurately weighing a certain amount of freeze-dried fig callus, adding liquid nitrogen according to 10g/500mL (precooling with liquid nitrogen before using a mortar, and adding sample amount not exceeding one third of the volume of the mortar), fully grinding, sieving with a 200-mesh sieve, and adding 50% ethanol according to a (m/v) feed liquid ratio of 1:10. Repeatedly extracting for 3 times at room temperature of 20-23-25deg.C and for 30min under the condition of 1300W ultrasonic power, filtering and mixing the extractive solutions, collecting clear supernatant, distilling under reduced pressure by rotary evaporator to remove ethanol (rotary evaporator condition: 32-38deg.C, rotation speed 60-80 rpm), and finally obtaining crude extract of fructus fici callus, and storing at 4deg.C or-20deg.C in refrigerator in dark place.
And (3) measuring the flavone content:
full wavelength scanning of rutin standard: 0.5mL (0.2 mg/mL) rutin standard 25mL volumetric flask, 1mL5% NaNO 2 (0.5 g sodium nitrite+9.5g ionized water), shaking and standing for 6min, adding 1mL of 10% Al (NO) 3 ) 3 (1 g of aluminum nitrate+46.8mL of water), shaking and standing for 6min, adding 10mL of 4% NaOH (3 g of sodium hydroxide+72 mL of water), adding 50% ethanol for constant volume, shaking and standing for 20min, scanning at full wavelength (400-600 nm, sampling interval of 1 nm), and determining the optimal absorbance.
Determination of total flavonoids: dissolving rutin standard 10mg in 50mL volumetric flask, adding 50% ethanol, fixing volume, placing 0.5, 1.0, 2.0, 3.0, 4.0, 5.0, 6.0mL into 25mL volumetric flask, respectively adding 50% ethanol to 10mL (9.5, 9, 8, 7, 6, 5, 4 mL), adding 1mL5% NaNO 2 (0.5 g sodium nitrite+9.5g ionized water), shaking and standing for 6min, adding 1mL of 10% Al (NO) 3 ) 3 (1 g of aluminum nitrate+46.8mL of water), shaking and standing for 6min, adding 10mL of 4% NaOH (3 g of sodium hydroxide+72 mL of water), adding 50% ethanol for constant volume, shaking and standing. And measuring the absorbance at 510nm to prepare the standard curve.
The total flavone solution (XmL or Xg) was added to a 50mL volumetric flask, 50% absolute ethanol was added to a constant volume, 0.5mL of the total flavone solution was added to a 25mL volumetric flask (0.5 mL of 50% ethanol was used as a blank), and absorbance was measured according to the above-mentioned measurement procedure.
Flavone extraction amount (mg/g) =total flavone concentration total volume of extract dilution fold/dry sample mass. See in particular table 3.
And (3) measuring polyphenol content:
gallic acid standard solution: gallic acid 50mg is weighed and fully dissolved in 10mL absolute ethyl alcohol, and distilled water is fixed to 100mL. Taking 0, 0.5, 1.0, 2.0, 3.0, 4.0 and 5.0mL respectively to fix the volume in a 25mL volumetric flask. Respectively taking 1.0mL of gallic acid standard solution with different mass concentrations, adding 5mL of water and 1mL of Fu Lin Fen reagent, shaking and mixing uniformly for 8min3ml of 7.5% Na was added 2 CO 3 (7.5 g of sodium carbonate+100 mL of water) was reacted in the dark for 2 hours, and the absorbance was measured at 765 nm.
Taking 1mL of a diluted sample, adding 5mL of water and 1mL of LFC reagent, uniformly mixing and vibrating, adding 3mL of 7.5% Na2CO3 for light-shielding reaction for 2h within 8min, measuring the absorbance value at 765nm wavelength, and substituting the absorbance value into the standard curve.
Flavone extraction amount (mg/g) =concentration calculated from standard curve (mg/g) ×volume of sample extract after dilution (ml)/dry weight of raw material (g). See in particular table 3.
TABLE 3 influence of different hormone concentration ratios on Ficus carica callus active ingredient
As can be seen from Table 3, experiments 1-4 show that, as the culture time of the fig callus increases, different addition amounts of zeatin have obvious influence on the contents of flavone and polyphenol, and after 25 days of culture, the contents of total phenol and flavone show obvious increasing trend, which is obviously higher than the different proportions of the same hormone. Experiments 9-12 show that the contents of flavone and polyphenol show a trend of increasing and then decreasing along with the increase of the culture time of the fig callus, and experiments 10-12 can obtain high-content active ingredients. Experiments 13-16 show that the contents of flavone and polyphenol show a trend of decreasing before increasing along with the increase of the culture time of the fig callus, and experiments 13 and 16 can obtain higher contents of active ingredients.
Experimental example 3 callus growth Condition
TABLE 4 influence of different hormone concentration ratios on Ficus carica callus appearance
As shown in tables 4 and 3, there was no direct connection between the growth rate and the content of the active ingredient, and as shown in tables 3 and 2, there was no direct connection between the content of the active ingredient and the weight gain of the callus.
The invention provides a culture method capable of obtaining high-content active substances, which selects a plurality of cytokinins to carry out multiplication culture of callus, and the ingredients are synergistic, compared with a plurality of fig multiplication medium formulas, the fresh weight of the callus obtained in one multiplication period can be increased by 5.52 times at most, and the content of active ingredients in the callus is obviously increased. Furthermore, the scheme can also omit seasons or plant reproduction periods, and fresh plant callus can be obtained at any time.
The above specific embodiments are provided for illustrative purposes only and are not intended to limit the invention, and modifications, no inventive contribution, will be made to the embodiments by those skilled in the art after having read the present specification, as long as they are within the scope of the patent statutes.
Claims (10)
1. The proliferation culture method of the fig callus is characterized by comprising the following steps of:
s1, callus induction: inoculating the explant on an induction culture medium, and performing dark culture for 20-30 days at the temperature of 23-25 ℃ to obtain callus;
s2, callus proliferation: spreading the callus obtained by culturing in the step S1 on a proliferation culture medium, and culturing for 25-35 days;
wherein the proliferation culture medium consists of a basic culture medium and a plant growth regulator; the basic culture medium comprises 4.41g/L of MS culture medium powder, 25-30g/L of sucrose and 6-8g/L of agar;
the plant growth regulator comprises one or more of the following components: 1-naphthylacetic acid 0.05-0.1mg/L, zeatin 3.7-4.3mg/L, 6-benzylaminopurine 3.7-8.3mg/L and thidiazuron 0.7-4.3mg/L.
2. The proliferation culture method according to claim 1, wherein the proliferation medium consists of a minimal medium and a plant growth regulator; the basic culture medium comprises 4.41g/L of MS culture medium powder, 25-30g/L of sucrose and 6-8g/L of agar, and the plant growth regulator comprises 0.1mg/L of 1-naphthylacetic acid and 3.7-4.3mg/L of zeatin.
3. The proliferation culture method according to claim 1, wherein the proliferation medium consists of a minimal medium and a plant growth regulator; the basic culture medium comprises 4.41g/L of MS culture medium powder, 25-30g/L of sucrose and 6-8g/L of agar, and the plant growth regulator comprises 3.7-4.3mg/L of 6-benzylaminopurine and 0.1mg/L of 1-naphthylacetic acid.
4. The proliferation culture method according to claim 1, wherein the proliferation medium consists of a minimal medium and a plant growth regulator; the basic culture medium comprises 4.41g/L of MS culture medium powder, 25-30g/L of sucrose and 6-8g/L of agar, and the plant growth regulator comprises 5.7-6.3mg/L of 6-benzylaminopurine and 0.1mg/L of 1-naphthylacetic acid.
5. The proliferation culture method according to claim 1, wherein the proliferation medium consists of a minimal medium and a plant growth regulator; the basic culture medium comprises 4.41g/L of MS culture medium powder, 25-30g/L of sucrose and 6-8g/L of agar, and the plant growth regulator comprises 7.7-8.3mg/L of 6-benzylaminopurine and 0.1mg/L of 1-naphthylacetic acid.
6. The proliferation culture method according to claim 1, wherein the proliferation medium consists of a minimal medium and a plant growth regulator; the basic culture medium comprises 4.41g/L of MS culture medium powder, 25-30g/L of sucrose and 6-8g/L of agar, and the plant growth regulator comprises 0.05mg/L of 1-naphthylacetic acid, 3.7-4.7mg/L of 6-benzylaminopurine and 0.7-1.3mg/L of thidiazuron.
7. The proliferation culture method according to claim 1, wherein the proliferation medium consists of a minimal medium and a plant growth regulator; the basic culture medium comprises 4.41g/L of MS culture medium powder, 25-30g/L of sucrose and 6-8g/L of agar, and the plant growth regulator comprises 0.05mg/L of 1-naphthylacetic acid, 3.7-4.3mg/L of 6-benzylaminopurine and 3.7-4.3mg/L of thidiazuron.
8. The proliferation culture method according to claim 1, wherein the induction medium is formulated as follows: MS culture medium powder 4.41g/L, sucrose 25-30g/L, agar 6-8g/L, 6-benzyl aminopurine 1.5-2.5mg/L, 1-naphthylacetic acid 0.45-0.55mg/L.
9. The proliferation culture method of claim 8, wherein the induction medium is formulated by: after the MS culture medium powder, sucrose and agar are prepared, the pH is adjusted to 5.8-6, and 6-benzyl amino purine and 1-naphthylacetic acid are added when the temperature is reduced to 55-65 ℃ for sterilization.
10. The proliferation culture method according to any one of claims 1-9, wherein the explant is stem segments or leaves of fig, and the length of the explant is 8-12mm.
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| US20020160513A1 (en) * | 2001-02-06 | 2002-10-31 | Hiroshima University | Cultured cells of Ficus stipulata Thunb. (= Ficus thunbergii) and a method for culturing tissues of the Ficus stipulata Thunb. By using said cultured cells |
| CN103299901A (en) * | 2013-05-15 | 2013-09-18 | 江苏丘陵地区镇江农业科学研究所 | In-vitro rapid proliferation method of Masui dauphine fig |
| CN103518625A (en) * | 2013-11-01 | 2014-01-22 | 重庆文理学院 | Tissue culture medium and in-vitro regeneration method for ficus pandurata blade |
| CN113557961A (en) * | 2021-08-19 | 2021-10-29 | 江苏农林职业技术学院 | Method for culturing fig callus |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20020160513A1 (en) * | 2001-02-06 | 2002-10-31 | Hiroshima University | Cultured cells of Ficus stipulata Thunb. (= Ficus thunbergii) and a method for culturing tissues of the Ficus stipulata Thunb. By using said cultured cells |
| CN103299901A (en) * | 2013-05-15 | 2013-09-18 | 江苏丘陵地区镇江农业科学研究所 | In-vitro rapid proliferation method of Masui dauphine fig |
| CN103518625A (en) * | 2013-11-01 | 2014-01-22 | 重庆文理学院 | Tissue culture medium and in-vitro regeneration method for ficus pandurata blade |
| CN113557961A (en) * | 2021-08-19 | 2021-10-29 | 江苏农林职业技术学院 | Method for culturing fig callus |
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