CN104472362A - Callus induction and anti-browning method for dracaena cochinchinensis - Google Patents
Callus induction and anti-browning method for dracaena cochinchinensis Download PDFInfo
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Abstract
The invention discloses a callus induction and anti-browning method for dracaena cochinchinensis. The method comprises the following steps: (1) using dracaena cochinchinensis seeds as explants for sterilizing; (2) putting the sterilized explants into an MS minimal medium for performing induced budding; (3) putting germ-free shoots into the MS minimal medium for culturing to obtain callus; (4) putting the callus into an MS enrichment medium for culturing to obtain a large amount of callus; and (5) putting the large amount of callus into an MS anti-browning culture medium for culturing to obtain the callus with fluffy texture. The callus obtained by using the method is viridis or cream yellow, high in growing speed and difficult to brown, and can produce a large amount of callus with good growth trend and low browning rate by means of induced proliferation; and therefore, high-quality materials are provided for extracting dragon's blood in medical science, and the market requirement can be further met.
Description
Technical field
The present invention relates to callus induction and the anti-browning method of the induction of a kind of plant callus and anti-browning method, particularly a kind of swordleaf dragon tree.
Background technology
Swordleaf dragon tree (Dracaena cochinchinensis (Lour) S.C.Chen) have another name called thousand wood, dragon's blood tree, toe dragon tree, be vulnerable species, belong to Chinese Second Class Key Protected Plant, be mainly distributed in the ground such as Guangxi, Yunnan, Hainan.Swordleaf dragon tree has very high medical value, and the dragon's blood extracted from its resin is called as Sanguis Draxonis, is China's tradition rare traditional Chinese medicine, has the use history of more than 1500 in China.In addition, its moulding is simple and tasteful, has very high ornamental value.The international nature of 2003 Nian Bei is classified as red list with the natural resources protection federation species incorporated into as being subject to endanger, and being decided to be the endangered species in the Red Data Book of Cape Verde, is the Original plant of current domestic generally acknowledged domestic Resina Draconis.
Record according to Compendium of Material Medica, Resina Draconis is warm in nature, flat, and taste is sweet, salty, nontoxic, enters blood system, returns lung, spleen, kidney three warp, has promoting blood circulation and removing blood stasis, swelling and pain relieving, astringing to arrest bleeding, softening and resolving hard mass, the remarkable efficacy such as regenerating tissue to heal wond, have the good reputation of " panacea of invigorating blood circulation ".Modern medicine study confirm, dragon's blood have improve body microcirculation, adjustment body metabolism, improve the effects such as body's immunity.Along with the expansion of dragon's blood range of application in clinical medicine, dragon's blood has the trend that supply falls short of demand.Add the growth of dragon tree slowly; within 1 year, trunk increasing is slightly less than 1 centimetre; Resina Draconis is again the red liquid of the injured rear outflow of dragon tree; output is very limited; current price has increased to 1000 ~ 1300 yuan/kg; cause people to implement irrational predatoriness to swordleaf dragon tree wild resource to fell, its wild resource is increasingly exhausted, extinction in imminent danger.Also the mixed adulterant having occurred multiple Resina Draconis thus throughout the country market circulates extensively, and has caused the market phenomenon of vicious circle and a lot of intoxication accident.Medicinal material swordleaf dragon tree is also owing to being subject to the impact of the various factors such as weather, the place of production, and quality of medicinal material has more significant difference.
Swordleaf dragon tree achieves preliminary progress on group culturation rapid propagating technology, but in explant propagation, induction just differentiation and again in atomization, autologous tissue can discharge brown material to medium and cause medium browning gradually, thus the quality having a strong impact on callus and aseptic seedling is even dead, serious restriction swordleaf dragon tree callus tissue culture efficiency, is difficult to the needs meeting large-scale production.For this reason, explore best hormonal factors and the condition of swordleaf dragon tree callus induction, and find better Browning control way by adding the mode such as anti-browning material, change culture environment, to effectively reducing melting brown rate, set up high-quality swordleaf dragon tree callus tissue culture system.
Summary of the invention
The object of this invention is to provide a kind of callus induction of swordleaf dragon tree and the method for anti-browning, it can produce a large amount of callus grown fine the short time, provide high-quality " dragon's blood " raw material for market.
The present invention achieves the above object by the following technical programs: a kind of callus induction of swordleaf dragon tree and the method for anti-browning, comprise the following steps:
(1) sterilization of explant: get swordleaf dragon tree seed as explant, first use liquid detergent aqueous cleaning the surface of the seed dirt, be placed in beaker and carry out running water 5 ~ 8min, then move on superclean bench, with 75% alcohol, seed is soaked 30S, aseptic water washing 1 time, then be 0.1%HgC1 by the 100ml concentration that with the addition of 1-2 and drip Tween-20
2sterilization 8 ~ 15min, sterile water soaks 3 times, inoculates after blotting explant surface moisture with aseptic filter paper, and wherein sterile water is through autoclaved distilled water;
(2) Seed inducement: be inoculated in MS inducing culture by the explant that step (1) obtains is 25 ± 2 DEG C in cultivation temperature, intensity of illumination 20 ~ 30 μm of ol/ (m
2s), light application time is cultivate 15d under the condition of 12h/d, seed sprouting, described Seed inducement MS inducing culture is methyl α-naphthyl acetate NAA, 30g/L sucrose of 6-benzyladenine 6-BA, the 0.1mg/L adding 0.5mg/L in MS minimal medium, the agar of 4.5g/L, the pH value of medium is 5.8;
(3) bud inducement obtains callus: the aseptic bud obtained in step (2) is placed in MS inducing culture, cultivation temperature 25 ± 2 DEG C, and intensity of illumination 20 ~ 30 μm of ol/ (m
2s), light application time is cultivate 7d under the condition of 10h/d to start to sprout, 15d just forms callus, described bud inducement MS inducing culture is add the 6-benzyladenine 6-BA of 0.1 ~ 2.0mg/L, the thiadiazole phenylurea TDZ of 0.05 ~ 0.5mg/L, the methyl α-naphthyl acetate NAA of 0.1 ~ 1.0mg/L, 2 of 0.1 ~ 1.0mg/L in MS minimal medium, 4-dichlorphenoxyacetic acid 2,4-D, 30g/L sucrose, the agar of 4.5g/L, the pH value of medium is 5.8;
(4) callus proliferation is cultivated: the callus obtained in step (3) is placed in MS proliferated culture medium, cultivation temperature 25 ± 2 DEG C, intensity of illumination 20 ~ 30 μm of ol/ (m
2s), MS proliferated culture medium is the 6-benzyladenine 6-BA, the methyl α-naphthyl acetate NAA of 0.5 ~ 2.0mg/L, the kinetin KT of 0.1 ~ 0.5mg/L that add 0.1 ~ 1.0mg/L in MS minimal medium, 30g/L sucrose, the agar of 4.5g/L, the pH value of medium is 5.8;
(5) anti-browning is cultivated: the callus obtained in step (4) is placed in MS anti-browning medium, cultivation temperature 25 ± 2 DEG C, and intensity of illumination 20 ~ 30 μm of ol/ (m
2s), light application time is cultivate under the condition of 10h/d to cultivate 40d respectively, and MS anti-browning medium is the VC adding anti-brown additive 20 ~ 100mg/L in MS minimal medium, the active carbon of 0.5 ~ 2.0g/L, 30g/L sucrose, the agar of 4.5g/L, the pH value of medium is 5.8;
Outstanding advantages of the present invention is:
(1) the present invention adopts biological technique method to carry out callus induction and anti-browning test to swordleaf dragon tree, carry out Fiber differentiation sprout by getting swordleaf dragon tree seed, cultivate the swordleaf dragon tree callus grown fine in a large number at short notice, realize the sustainable use to its herb resource.
(2) the present invention relates to 6-benzyladenine 6-BA, NAA, KT and 2,4-D can improve callus proliferation efficiency; Thiadiazole phenylurea TDZ is a kind of biological regulator, and it can induce explant to occur from Callus formation to somatic embryo, and VC and active carbon can Browning controls.Above chemicalss etc. are cheap, and cost is low.
(3) the swordleaf dragon tree callus tissue culture 30d rate of increase adopting cultural method of the present invention to obtain reaches 64.3%, peak green, and quality is loosened, and callus browning rate is 8.2%, obtains high-quality callus.
Embodiment
Below in conjunction with embodiment, technical scheme of the present invention is further illustrated.
Embodiment 1
(1) sterilization of explant: get swordleaf dragon tree seed as explant, first use liquid detergent aqueous cleaning the surface of the seed dirt, be placed in beaker and carry out running water 5 ~ 8min, then move on superclean bench, with 75% alcohol, seed is soaked 30S, aseptic water washing 1 time, then be 0.1%HgC1 by the 100ml concentration that with the addition of 1-2 and drip Tween-20
2sterilization 8 ~ 15min, sterile water soaks 3 times, inoculates after blotting explant surface moisture with aseptic filter paper, and wherein sterile water is through autoclaved distilled water;
(2) Seed inducement: be inoculated in MS inducing culture by the explant that step (1) obtains is 25 ± 2 DEG C in cultivation temperature, intensity of illumination 20 ~ 30 μm of ol/ (m
2s), light application time is cultivate 15d under the condition of 12h/d, seed sprouting, described Seed inducement MS inducing culture is methyl α-naphthyl acetate NAA, 30g/L sucrose of 6-benzyladenine 6-BA, the 0.1mg/L adding 0.5mg/L in MS minimal medium, the agar of 4.5g/L, the pH value of medium is 5.8.
(3) bud inducement obtains callus: the aseptic bud obtained in step (2) is placed in MS inducing culture, cultivation temperature 25 ± 2 DEG C, and intensity of illumination 20 ~ 30 μm of ol/ (m
2s), light application time is cultivate 9d under the condition of 10h/d to start to sprout, 18d just forms callus, described bud inducement MS inducing culture is 2, the 4-dichlorphenoxyacetic acids 2,4-D of methyl α-naphthyl acetate NAA, 0.1mg/L of thiadiazole phenylurea TDZ, the 0.1mg/L of 6-benzyladenine 6-BA, the 0.2mg/L adding 0.1mg/L in MS minimal medium, 30g/L sucrose, the agar of 4.5g/L, the pH value of medium is 5.8, and callus induction rate is 51.9%;
(4) callus proliferation is cultivated: the callus obtained in step (3) is placed in MS proliferated culture medium, cultivation temperature 25 ± 2 DEG C, intensity of illumination 20 ~ 30 μm of ol/ (m
2s), MS proliferated culture medium is the kinetin KT of methyl α-naphthyl acetate NAA, the 0.1mg/L of 6-benzyladenine 6-BA, the 0.5mg/L adding 0.1mg/L in MS minimal medium, 30g/L sucrose, the agar of 4.5 g/L, the pH value of medium is 5.8, and callus proliferation rate is 49.7%;
(5) anti-browning is cultivated: the callus obtained in step (4) is placed in MS anti-browning medium, cultivation temperature 25 ± 2 DEG C, and intensity of illumination 20 ~ 30 μm of ol/ (m
2s), light application time is cultivate under the condition of 10h/d to cultivate 40d respectively, and anti-browning medium is the VC adding anti-brown additive 20mg/L in MS minimal medium, the active carbon of 0.5g/L, 30g/L sucrose, the agar of 4.5g/L, the pH value of medium is 5.8, and callus browning rate is 31.3%;
Embodiment 2
(1) sterilization of explant: get swordleaf dragon tree seed as explant, first use liquid detergent aqueous cleaning the surface of the seed dirt, be placed in beaker and carry out running water 5 ~ 8min, then move on superclean bench, with 75% alcohol, seed is soaked 30S, aseptic water washing 1 time, then be 0.1%HgC1 by the 100ml concentration that with the addition of 1-2 and drip Tween-20
2sterilization 8 ~ 15min, sterile water soaks 3 times, inoculates after blotting explant surface moisture with aseptic filter paper, and wherein sterile water is through autoclaved distilled water;
(2) Seed inducement: be inoculated in MS inducing culture by the explant that step (1) obtains is 25 ± 2 DEG C in cultivation temperature, intensity of illumination 20 ~ 30 μm of ol/ (m
2s), light application time is cultivate 15d under the condition of 12h/d, seed sprouting, described Seed inducement MS inducing culture is methyl α-naphthyl acetate NAA, 30g/L sucrose of 6-benzyladenine 6-BA, the 0.1mg/L adding 0.5mg/L in MS minimal medium, the agar of 4.5g/L, the pH value of medium is 5.8.
(3) bud inducement obtains callus: the aseptic bud obtained in step (2) is placed in MS inducing culture, cultivation temperature 25 ± 2 DEG C, and intensity of illumination 20 ~ 30 μm of ol/ (m
2s), light application time is cultivate 7d under the condition of 10h/d to start to sprout, 16d just forms callus, described bud inducement MS inducing culture is 2, the 4-dichlorphenoxyacetic acids 2,4-D of methyl α-naphthyl acetate NAA, the 1.0mg/L of thiadiazole phenylurea TDZ, the 0.5mg/L adding 1.0mg/L 6-benzyladenine 6-BA, 0.05mg/L in MS minimal medium, 30g/L sucrose, the agar of 4.5g/L, the pH value of medium is 5.8, and callus induction rate is 46.2%;
(4) callus proliferation is cultivated: the callus obtained in step (3) is placed in MS proliferated culture medium, cultivation temperature 25 ± 2 DEG C, intensity of illumination 20 ~ 30 μm of ol/ (m
2s), MS proliferated culture medium is the kinetin KT of methyl α-naphthyl acetate NAA, the 0.5mg/L of 6-benzyladenine 6-BA, the 1.0mg/L adding 0.5mg/L in MS minimal medium, 30g/L sucrose, the agar of 4.5g/L, the pH value of medium is 5.8, and callus proliferation rate is 58.3%;
(5) anti-browning is cultivated: the callus obtained in step (4) is placed in MS anti-browning medium, cultivation temperature 25 ± 2 DEG C, and intensity of illumination 20 ~ 30 μm of ol/ (m
2s), light application time is cultivate under the condition of 10h/d to cultivate 40d respectively, and anti-browning medium is the VC adding anti-brown additive 100mg/L in MS minimal medium, the active carbon of 1.2g/L, 30g/L sucrose, the agar of 4.5g/L, the pH value of medium is 5.8, and callus browning rate is 8.2%;
Embodiment 3
(1) sterilization of explant: get swordleaf dragon tree seed as explant, first use liquid detergent aqueous cleaning the surface of the seed dirt, be placed in beaker and carry out running water 5 ~ 8min, then move on superclean bench, with 75% alcohol, seed is soaked 30S, aseptic water washing 1 time, then be 0.1%HgC1 by the 100ml concentration that with the addition of 1-2 and drip Tween-20
2sterilization 8 ~ 15min, sterile water soaks 3 times, inoculates after blotting explant surface moisture with aseptic filter paper, and wherein sterile water is through autoclaved distilled water;
(2) Seed inducement: be inoculated in MS inducing culture by the explant that step (1) obtains is 25 ± 2 DEG C in cultivation temperature, intensity of illumination 20 ~ 30 μm of ol/ (m
2s), light application time is cultivate 15d under the condition of 12h/d, seed sprouting, described Seed inducement MS inducing culture is methyl α-naphthyl acetate NAA, 30g/L sucrose of 6-benzyladenine 6-BA, the 0.1mg/L adding 0.5mg/L in MS minimal medium, the agar of 4.5g/L, the pH value of medium is 5.8.
(3) bud inducement obtains callus: the aseptic bud obtained in step (2) is placed in MS inducing culture, cultivation temperature 25 ± 2 DEG C, and intensity of illumination 20 ~ 30 μm of ol/ (m
2s), light application time is cultivate 7d under the condition of 10h/d to start to sprout, 15d just forms callus, described bud inducement MS inducing culture is 2, the 4-dichlorphenoxyacetic acids 2,4-D of methyl α-naphthyl acetate NAA, 0.3mg/L of thiadiazole phenylurea TDZ, the 1.0mg/L of 6-benzyladenine 6-BA, the 0.2mg/L adding 1.0mg/L in MS minimal medium, 30g/L sucrose, the agar of 4.5g/L, the pH value of medium is 5.8, and callus induction rate is 95.8%;
(4) callus proliferation is cultivated: the callus obtained in step (3) is placed in MS proliferated culture medium, cultivation temperature 25 ± 2 DEG C, and intensity of illumination 20 ~ 30 μm of ol/ (m
2s), MS proliferated culture medium is the kinetin KT of methyl α-naphthyl acetate NAA, the 0.2mg/L of 6-benzyladenine 6-BA, the 2.0mg/L adding 0.5mg/L in MS minimal medium, 30g/L sucrose, the agar of 4.5g/L, the pH value of medium is 5.8, and callus proliferation rate is 62.5%;
(5) anti-browning is cultivated: the callus obtained in step (4) is placed in MS anti-browning medium, cultivation temperature 25 ± 2 DEG C, and intensity of illumination 20 ~ 30 μm of ol/ (m
2s), light application time is cultivate under the condition of 10h/d to cultivate 40d respectively, anti-browning medium is the VC adding anti-brown additive 100mg/L in MS minimal medium, the active carbon of 2.0g/L, 30g/L sucrose, the agar of 4.5g/L, the pH value of medium is 5.8, and callus browning rate is 22.4%;
Embodiment 4
(1) sterilization of explant: get swordleaf dragon tree seed as explant, first use liquid detergent aqueous cleaning the surface of the seed dirt, be placed in beaker and carry out running water 5 ~ 8min, then move on superclean bench, with 75% alcohol, seed is soaked 30S, aseptic water washing 1 time, then be 0.1%HgC1 by the 100ml concentration that with the addition of 1-2 and drip Tween-20
2sterilization 8 ~ 15min, sterile water soaks 3 times, inoculates after blotting explant surface moisture with aseptic filter paper, and wherein sterile water is through autoclaved distilled water;
(2) Seed inducement: be inoculated in MS inducing culture by the explant that step (1) obtains is 25 ± 2 DEG C in cultivation temperature, intensity of illumination 20 ~ 30 μm of ol/ (m
2s), light application time is cultivate 15d under the condition of 12h/d, seed sprouting, described Seed inducement MS inducing culture is methyl α-naphthyl acetate NAA, 30g/L sucrose of 6-benzyladenine 6-BA, the 0.1mg/L adding 0.5mg/L in MS minimal medium, the agar of 4.5g/L, the pH value of medium is 5.8.
(3) bud inducement obtains callus: the aseptic bud obtained in step (2) is placed in MS inducing culture, cultivation temperature 25 ± 2 DEG C, and intensity of illumination 20 ~ 30 μm of ol/ (m
2s), light application time is cultivate 8d under the condition of 10h/d to start to sprout, 17d just forms callus, described bud inducement MS inducing culture is 2, the 4-dichlorphenoxyacetic acids 2,4-D of methyl α-naphthyl acetate NAA, 0.3mg/L of thiadiazole phenylurea TDZ, the 0.5mg/L of 6-benzyladenine 6-BA, the 0.5mg/L adding 2.0mg/L in MS minimal medium, 30g/L sucrose, the agar of 4.5g/L, the pH value of medium is 5.8, and callus induction rate is 78.5%;
(4) callus proliferation is cultivated: the callus obtained in step (3) is placed in MS proliferated culture medium, cultivation temperature 25 ± 2 DEG C, intensity of illumination 20 ~ 30 μm of ol/ (m
2s), MS proliferated culture medium is the kinetin KT of methyl α-naphthyl acetate NAA, the 0.2mg/L of 6-benzyladenine 6-BA, the 1.0mg/L adding 1.0mg/L in MS minimal medium, 30g/L sucrose, the agar of 4.5g/L, the pH value of medium is 5.8, and callus proliferation rate is 64.3%;
(5) anti-browning is cultivated: the callus obtained in step (4) is placed in MS anti-browning medium, cultivation temperature 25 ± 2 DEG C, and intensity of illumination 20 ~ 30 μm of ol/ (m
2s), light application time is cultivate under the condition of 10h/d to cultivate 40d respectively, and anti-browning medium is the VC adding anti-brown additive 50mg/L in MS minimal medium, the active carbon of 1.2g/L, the agar of 30g/L sucrose and 4.5g/L, the pH value of medium is 5.8, and melting brown rate is 19.5%.
Claims (1)
1. the callus induction of swordleaf dragon tree and a method for anti-browning, is characterized in that: the method comprises the following steps:
(1) sterilization of explant: get swordleaf dragon tree seed as explant, first use liquid detergent aqueous cleaning the surface of the seed dirt, be placed in beaker and carry out running water 5 ~ 8min, then move on superclean bench, with 75% alcohol, seed is soaked 30S, aseptic water washing 1 time, then be 0.1%HgC1 by the 100ml concentration that with the addition of 1-2 and drip Tween-20
2sterilization 8 ~ 15min, sterile water soaks 3 times, inoculates after blotting explant surface moisture with aseptic filter paper, and wherein sterile water is through autoclaved distilled water;
(2) Seed inducement: be inoculated in MS inducing culture by the explant that step (1) obtains is 25 ± 2 DEG C in cultivation temperature, intensity of illumination 20 ~ 30 μm of ol/ (m
2s), light application time is cultivate 15d under the condition of 12h/d, seed sprouting, described Seed inducement MS inducing culture is methyl α-naphthyl acetate NAA, 30g/L sucrose of 6-benzyladenine 6-BA, the 0.1mg/L adding 0.5mg/L in MS minimal medium, the agar of 4.5g/L, the pH value of medium is 5.8;
(3) bud inducement obtains callus: the aseptic bud obtained in step (2) is placed in MS inducing culture, cultivation temperature 25 ± 2 DEG C, and intensity of illumination 20 ~ 30 μm of ol/ (m
2s), light application time is cultivate 7d under the condition of 10h/d to start to sprout, 15d just forms callus, described bud inducement MS inducing culture is add the 6-benzyladenine 6-BA of 0.1 ~ 2.0mg/L, the thiadiazole phenylurea TDZ of 0.05 ~ 0.5mg/L, the methyl α-naphthyl acetate NAA of 0.1 ~ 1.0mg/L, 2 of 0.1 ~ 1.0mg/L in MS minimal medium, 4-dichlorphenoxyacetic acid 2,4-D, 30g/L sucrose, the agar of 4.5g/L, the pH value of medium is 5.8;
(4) callus proliferation is cultivated: the callus obtained in step (3) is placed in MS proliferated culture medium, cultivation temperature 25 ± 2 DEG C, intensity of illumination 20 ~ 30 μm of ol/ (m
2s), MS proliferated culture medium is the 6-benzyladenine 6-BA, the methyl α-naphthyl acetate NAA of 0.5 ~ 2.0mg/L, the kinetin KT of 0.1 ~ 0.5mg/L that add 0.1 ~ 1.0mg/L in MS minimal medium, 30g/L sucrose, the agar of 4.5g/L, the pH value of medium is 5.8;
(5) anti-browning is cultivated: the callus obtained in step (4) is placed in MS anti-browning medium, cultivation temperature 25 ± 2 DEG C, and intensity of illumination 20 ~ 30 μm of ol/ (m
2s), light application time is cultivate under the condition of 10h/d to cultivate 40d respectively, and MS anti-browning medium is the VC adding anti-brown additive 20 ~ 100mg/L in MS minimal medium, the active carbon of 0.5 ~ 2.0g/L, 30g/L sucrose, the agar of 4.5g/L, the pH value of medium is 5.8.
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Cited By (3)
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| CN105340736A (en) * | 2015-11-09 | 2016-02-24 | 广西壮族自治区药用植物园 | Dracaena cochinchinensis loose callus induction method |
| CN115280986A (en) * | 2022-07-28 | 2022-11-04 | 河南科技大学 | Method for rapid propagation of seedlings by cutting with radix paeoniae alba root segments |
| CN115968787A (en) * | 2023-02-20 | 2023-04-18 | 广西农业职业技术大学 | Method for reducing browning of kidney bean in-vitro regeneration system |
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| CN105340736A (en) * | 2015-11-09 | 2016-02-24 | 广西壮族自治区药用植物园 | Dracaena cochinchinensis loose callus induction method |
| CN115280986A (en) * | 2022-07-28 | 2022-11-04 | 河南科技大学 | Method for rapid propagation of seedlings by cutting with radix paeoniae alba root segments |
| CN115968787A (en) * | 2023-02-20 | 2023-04-18 | 广西农业职业技术大学 | Method for reducing browning of kidney bean in-vitro regeneration system |
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