CN103436469A - Propagation expanding method of medicinal ferula asafetida endophyte - Google Patents
Propagation expanding method of medicinal ferula asafetida endophyte Download PDFInfo
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- CN103436469A CN103436469A CN2013103532458A CN201310353245A CN103436469A CN 103436469 A CN103436469 A CN 103436469A CN 2013103532458 A CN2013103532458 A CN 2013103532458A CN 201310353245 A CN201310353245 A CN 201310353245A CN 103436469 A CN103436469 A CN 103436469A
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Abstract
The invention discloses a propagation expanding method of medicinal ferula asafetida endophyte. The method is mainly implemented by preparing explant, isolating and culturing fungal, isolating and culturing bacteria, and isolating and culturing actinomycetes. The method is to isolate ferula asafetida endophyte, especially culture and carry out propagation expanding on the ferula asafetida endophyte, so as to further develop and utilize the medicinal original plant of ferula asafetida to provide the ferula asafetida endophyte with adequate quantity and sustainable utilization, and recover the ecological resources of the ferula asafetida original plant. Therefore, the method is of great significance.
Description
Technical field
The present invention relates to a kind of expanding propagation method of medicinal asafoetide endophyte.
Background technology
Ubiquity endophyte in plant materials, because it is lived in and be there is no the health plant of external infection symptoms organization internal, so the existence of endophyte of plant and effect undiscovered for a long time.
Until because livestock has eaten the herbage that infects endogenetic fungus, cause heavy losses to livestock industry the thirties in 20th century, just starting has had Preliminary study to endophyte of plant.
Endophyte of plant, as the important component part of plant microecosystem, in long-term coevolution process, has formed the relations such as mutual reciprocity and mutual benefit or harm plant-growth with plant.Most of endophyte has and strengthens host's effect of the aspects such as synthetic (or self having synthetic certain compound ability) of some effective active compositions in stress tolerance, the growth that promotes host plant and the host of environment to external world, and further investigation may and improve medicinal plant output etc. to the artificial growth of medicinal plant and have vital role.
Asafoetide bitter, suffering, warm in nature, return spleen, stomach warp, the effect that disappear long-pending, loose ruffian, desinsection are arranged, it is the traditional Chinese medicine of China, being also the distinctive rare medicinal material in Xinjiang, being incorporated into and going through one one of edition Pharmacopoeia of the People's Republic of China, is the resin that Ji Yuan is Ferula sinkiangensisK.M.Shen (Ferula sinkiangensis K.M. Shen) or Ferula fukanensis K.M.Shen (Ferufukanensis K.M. Shen).
Because of the restriction of the aspects such as medicinal asafoetide plant self physiology reproduction and unauthorized and excessive mining for a long time, its resource is suffered considerable damage at present, endangered, and is laid special stress on protecting threatened plant by three grades.
Medicinal asafoetide endophyte mainly contain fungi, bacterium and and actinomycetes, the secondary metabolite of these microorganisms and being studied with dependency of Ferula plant-growth etc., have great importance with the ecological resources of asafoetide to restorative.
And in real application research, need to use in large quantities the endophyte thalline, and the cultivation of thalline needs again a longer process, the medicinal former plant of asafoetide is ephemeroid in perennial hapanthous early spring, contain abundant natural gum, but, along with strain is accumulated age year by year, these natural gum cause certain difficulty to the acquisition of endophyte, cause the famine of thalline in research, so the thalline deposit of some amount is related to normally carrying out of research.
Therefore, the asafoetide endophyte is separated, particularly to the cultivation of thalline with expand numerously, for the medicinal former plant that further develops asafoetide provides the thalline of sufficient amount, and then the ecological resources of the former plant of asafoetide for restorative, had great importance.
Summary of the invention
The expanding propagation method that the purpose of this invention is to provide a kind of medicinal asafoetide endophyte.
Purpose of the present invention is mainly by following process implementation:
Explant is made: get the root of fresh Ferula sinkiangensisK.M.Shen, Ferula fukanensis K.M.Shen, sheep food asafoetide as explant, be cut into the segment of 0.5～2cm, through the alcohol disinfecting of concentration 65～85% 0.5～1.5 minute, through the clorox of concentration 2～4%, sterilize 5～8 minutes again, sterilized water washing afterwards 3～5 times, draining the water is placed in 2～5 ℃ of environment and saves backup.
The separation of fungi and cultivation: by the explant of above-mentioned sterilization, be cut into 0.3～0.6cm fritter and be inoculated in substratum, each culture dish is preferably inoculated 3～7.
Fungi culture medium used is a kind of in wheat bran substratum, rose bengal medium.
At 23～27 ℃ of temperature, secretly cultivate 4～8 days, after mycelia grows, picking mycelia tip, be placed in PDA substratum purifying step by step, until form single bacterium colony.
Described purifying step by step is: impure bacterium colony picking edge mycelia is placed in to the culture dish purifying and is cultured to single bacterium colony, for unpurified bacterium colony, continue picking edge purifying until become single bacterium colony.
The separation of bacterium and cultivation: the asafoetide explant after the cancellation poison, homogenate, then being diluted to content with sterilized water by weight is 10
-3～10
-6diluent, insert in beef-protein medium, temperature be under 36～39 ℃ dark cultivate 24～48 hours after, the single bacterium colony of picking is streak culture step by step, until form single bacterium colony.
Described streak culture step by step: as impure bacterium colony to be chosen to single bacterium colony streak culture to single bacterium colony, for purifying bacterium colony not, continue to choose bacterium streak culture, until become single bacterium colony.
Actinomycetic separation and cultivation: the asafoetide explant after the cancellation poison, homogenate, then being diluted to content with sterilized water by weight is 10
-2～10
-5diluent, insert in the Gause I substratum dark the cultivation after 14～21 days, a small amount of mycelia in picking edge purifying is step by step cultivated, until form single bacterium colony.
Described purifying step by step is: impure bacterium colony picking edge mycelia is placed in to the culture dish purifying and is cultured to single bacterium colony, for unpurified bacterium colony, continue picking edge purifying until become single bacterium colony.
Above-mentioned bacterium and actinomycetic diluent are preferably prepared according to the following steps: the asafoetide explant after the cancellation poison, put into the mortar grinding of having sterilized and be pulpous state, by every 1 gram jelly, add 8～12 ml sterile waters, mix rear standingly more than 20 minutes, then therefrom draw supernatant liquid, add 8～12 ml sterile waters by 1 milliliter again, mix rear standingly more than 20 minutes, continue successively dilution, obtain required diluent.
If, in the situation that had the endophyte bacterial classification, can directly enter cultivation and expand numerous.
The present invention is separated the asafoetide endophyte, particularly to the cultivation of thalline with expand numerous, for the medicinal former plant that further develops asafoetide provides the thalline of sufficient amount and sustainable use, and then the ecological resources of the former plant of asafoetide for restorative, have great importance.
Embodiment
being formulated as follows of substratum.
Wheat bran substratum: more than wheat bran 250～350g is added in 1L water and boils 30min, get juice, add again glucose 150～250 g/L by juice, potassium primary phosphate 25～35 g/L, sal epsom 10～20 g/L, agar 100～150 g/L.
Rose bengal medium: peptone 4～6g, glucose 8～12g, potassium primary phosphate 0.5～1.5g, sal epsom (MgSO47H2O) 0.3～0.6g, agar 15～25g, 1/3000 rose-bengal solution 100mL, distilled water 1000mL, paraxin 0.05～0.15g.
The PDA substratum: 100～300g potato is cleaned, be cut into the bulk of 1～2 cm size, add in 1L water and boil 15～20min, filtered through gauze is got juice, add 15～25g glucose, be settled to 1000mL after 15～25g agar, the Streptomycin sulphate that adds 0.5～1.5g to time in 60 degree to be cooled, with anti-bacteria and actinomycetic growth; Purifying, when preservation, does not add Streptomycin sulphate.
Beef-protein medium: extractum carnis 2～4g, peptone 8～12g, Nacl5g, agar 15～20g, water 1000mL, adjust pH is to PH7.4～7.6.
Gause I substratum: by Zulkovsky starch 15～25g, saltpetre 0.5～1.5g, K
2hPO
40.4～0.6g, MgSO
47H
2o 0.4～0.6g, sodium-chlor 0.4～0.6g, ferrous sulfate 0.01～0.02g, agar 15～30g, dissolve in distilled water 1000 mL, then add potassium bichromate solution 2～6ml of 2～4%, adjust pH to 7.2～7.4.
embodiment 1:get the root of fresh Ferula sinkiangensisK.M.Shen as explant, be cut into the segment of 1cm, through the alcohol disinfecting of concentration 70% 1.5 minutes, then through the clorox sterilization of concentration 3% 5 minutes, sterilized water washing afterwards 3 times, draining the water is placed in 4 ℃ of environment and saves backup.
The separation of fungi and cultivation: by the explant of above-mentioned sterilization, be cut into the 0.3cm fritter and be inoculated in the wheat bran substratum, in each culture dish, inoculation is 5; At 25 ℃ of temperature, secretly cultivate 5 days, after mycelia grows, picking mycelia tip, be placed in PDA substratum purifying step by step, until form single bacterium colony.Described purifying step by step is: impure bacterium colony picking edge mycelia is placed in to the culture dish purifying and is cultured to single bacterium colony, for unpurified bacterium colony, continue picking edge purifying until become single bacterium colony.
The separation of bacterium and cultivation: the asafoetide explant after the cancellation poison, homogenate, then being diluted to content with sterilized water by weight is 10
-3～10
-6diluent, insert in beef-protein medium, temperature be under 38 ℃ dark cultivate 24 hours after, the single bacterium colony of picking is streak culture step by step, until form single bacterium colony.Described streak culture step by step: as impure bacterium colony to be chosen to single bacterium colony streak culture to single bacterium colony, for purifying bacterium colony not, continue to choose bacterium streak culture, until become single bacterium colony.
Actinomycetic separation and cultivation: the asafoetide explant after the cancellation poison, homogenate, then being diluted to content with sterilized water by weight is 10
-2～10
-5diluent, insert in the Gause I substratum dark the cultivation after 15 days, a small amount of mycelia in picking edge purifying is step by step cultivated, until form single bacterium colony.Described purifying step by step is: impure bacterium colony picking edge mycelia is placed in to the culture dish purifying and is cultured to single bacterium colony, for unpurified bacterium colony, continue picking edge purifying until become single bacterium colony.
embodiment 2:get the root of fresh Ferula sinkiangensisK.M.Shen, Ferula fukanensis K.M.Shen, sheep food asafoetide as explant, be cut into the segment of 1.5cm, through the alcohol disinfecting of concentration 75% 1.5 minutes, then through the clorox sterilization of concentration 2% 8 minutes, sterilized water washing afterwards 5 times, draining the water is placed in 5 ℃ of environment and saves backup.
The separation of fungi and cultivation: by the explant of above-mentioned sterilization, be cut into the 0.5cm fritter and be inoculated in the wheat bran substratum, 7 of each culture dish inoculations; At 23 ℃ of temperature, secretly cultivate 8 days, after mycelia grows, picking mycelia tip, be placed in PDA substratum purifying step by step, until form single bacterium colony.Described purifying step by step is: impure bacterium colony picking edge mycelia is placed in to the culture dish purifying and is cultured to single bacterium colony, for unpurified bacterium colony, continue picking edge purifying until become single bacterium colony.
The separation of bacterium and cultivation: the asafoetide explant after the cancellation poison, homogenate, then being diluted to content with sterilized water by weight is 10
-3～10
-6diluent, insert in beef-protein medium, temperature be under 39 ℃ dark cultivate 2 hours after, the single bacterium colony of picking is streak culture step by step, until form single bacterium colony.Described streak culture step by step: as impure bacterium colony to be chosen to single bacterium colony streak culture to single bacterium colony, for purifying bacterium colony not, continue to choose bacterium streak culture, until become single bacterium colony.
Actinomycetic separation and cultivation: the asafoetide explant after the cancellation poison, put into the mortar grinding of having sterilized and be pulpous state, by every 1 gram jelly, add 8～12 ml sterile waters, mix rear standingly more than 20 minutes, then therefrom draw supernatant liquid, add 8～12 ml sterile waters by 1 milliliter again, mix rear standingly more than 20 minutes, continue successively dilution, obtaining content is 10
-2～10
-5diluent, insert in the Gause I substratum dark the cultivation after 15 days, a small amount of mycelia in picking edge purifying is step by step cultivated, until form single bacterium colony.Described purifying step by step is: impure bacterium colony picking edge mycelia is placed in to the culture dish purifying and is cultured to single bacterium colony, for unpurified bacterium colony, continue picking edge purifying until become single bacterium colony.
embodiment 3:get the root of fresh Ferula sinkiangensisK.M.Shen, Ferula fukanensis K.M.Shen, sheep food asafoetide as explant, be cut into the segment of 2cm, through the alcohol disinfecting of concentration 80% 1 minute, then through the clorox sterilization of concentration 4% 5 minutes, sterilized water washing afterwards 3 times, draining the water is placed in 3 ℃ of environment and saves backup.
The separation of fungi and cultivation: by the explant of above-mentioned sterilization, be cut into the 0.5cm fritter and be inoculated in rose bengal medium, 4 of each culture dish inoculations; At 26 ℃ of temperature, secretly cultivate 6 days, after mycelia grows, picking mycelia tip, be placed in PDA substratum purifying step by step, until form single bacterium colony.Described purifying step by step is: impure bacterium colony picking edge mycelia is placed in to the culture dish purifying and is cultured to single bacterium colony, for unpurified bacterium colony, continue picking edge purifying until become single bacterium colony.
The separation of bacterium and cultivation: the asafoetide explant after the cancellation poison, put into the mortar grinding of having sterilized and be pulpous state, by every 1 gram jelly, add 8～12 ml sterile waters, mix rear standingly more than 20 minutes, then therefrom draw supernatant liquid, add 8～12 ml sterile waters by 1 milliliter again, mix rear standingly more than 20 minutes, continue successively dilution, obtaining content is 10
-3～10
-6diluent, insert in beef-protein medium, temperature be under 37 ℃ dark cultivate 36 hours after, the single bacterium colony of picking is streak culture step by step, until form single bacterium colony.Described streak culture step by step: as impure bacterium colony to be chosen to single bacterium colony streak culture to single bacterium colony, for purifying bacterium colony not, continue to choose bacterium streak culture, until become single bacterium colony.
Actinomycetic separation and cultivation: the asafoetide explant after the cancellation poison, put into the mortar grinding of having sterilized and be pulpous state, by every 1 gram jelly, add 8～12 ml sterile waters, mix rear standingly more than 20 minutes, then therefrom draw supernatant liquid, add 8～12 ml sterile waters by 1 milliliter again, mix rear standingly more than 20 minutes, continue successively dilution, obtaining content is 10
-4～10
-5diluent, insert in the Gause I substratum dark the cultivation after 20 days, a small amount of mycelia in picking edge purifying is step by step cultivated, until form single bacterium colony.Described purifying step by step is: impure bacterium colony picking edge mycelia is placed in to the culture dish purifying and is cultured to single bacterium colony, for unpurified bacterium colony, continue picking edge purifying until become single bacterium colony.
embodiment 4:get the root of fresh Ferula sinkiangensisK.M.Shen, Ferula fukanensis K.M.Shen, sheep food asafoetide as explant, be cut into the segment of 0.5cm, through the alcohol disinfecting of concentration 65% 1 minute, through the clorox of concentration 4%, sterilize 5 minutes again, sterilized water washing afterwards 3 times, drain to being placed on without dripping phenomenon in 2 ℃ of environment and save backup.
The separation of fungi and cultivation: by the explant of above-mentioned sterilization, be cut into the 0.5cm fritter and be inoculated in the wheat bran substratum, 6 of each culture dish inoculations; At 26 ℃ of temperature, secretly cultivate 5 days, after mycelia grows, picking mycelia tip, be placed in PDA substratum purifying step by step, until form single bacterium colony.Described purifying step by step is: impure bacterium colony picking edge mycelia is placed in to the culture dish purifying and is cultured to single bacterium colony, for unpurified bacterium colony, continue picking edge purifying until become single bacterium colony.
The separation of bacterium and cultivation: the asafoetide explant after the cancellation poison, homogenate, then being diluted to content with sterilized water by weight is 10
-3～10
-6diluent, insert in beef-protein medium, temperature be under 37 ℃ dark cultivate 24 hours after, the single bacterium colony of picking is streak culture step by step, until form single bacterium colony.Described streak culture step by step: as impure bacterium colony to be chosen to single bacterium colony streak culture to single bacterium colony, for purifying bacterium colony not, continue to choose bacterium streak culture, until become single bacterium colony.
Actinomycetic separation and cultivation: the asafoetide explant after the cancellation poison, homogenate, then being diluted to content with sterilized water by weight is 10
-3～10
-4diluent, insert in the Gause I substratum dark the cultivation after 18 days, a small amount of mycelia in picking edge purifying is step by step cultivated, until form single bacterium colony.Described purifying step by step is: impure bacterium colony picking edge mycelia is placed in to the culture dish purifying and is cultured to single bacterium colony, for unpurified bacterium colony, continue picking edge purifying until become single bacterium colony.
embodiment 5:get the root of fresh Ferula sinkiangensisK.M.Shen, Ferula fukanensis K.M.Shen, sheep food asafoetide as explant, be cut into the segment of 0.5cm, through the alcohol disinfecting of concentration 70% 0.5 minute, then through the clorox sterilization of concentration 3% 5 minutes, sterilized water washing afterwards 5 times, drain to without vast expense of water water, being placed in 4 ℃ of environment and saving backup.
The separation of fungi and cultivation: by the explant of above-mentioned sterilization, be cut into the 0.5cm fritter and be inoculated in rose bengal medium, 7 of each culture dish inoculations; At 25 ℃ of temperature, secretly cultivate 5 days, after mycelia grows, picking mycelia tip, be placed in PDA substratum purifying step by step, until form single bacterium colony.Described purifying step by step is: impure bacterium colony picking edge mycelia is placed in to the culture dish purifying and is cultured to single bacterium colony, for unpurified bacterium colony, continue picking edge purifying until become single bacterium colony.
The separation of bacterium and cultivation: the asafoetide explant after the cancellation poison, put into the mortar grinding of having sterilized and be pulpous state, by every 1 gram jelly, add 8～12 ml sterile waters, mix rear standingly more than 20 minutes, then therefrom draw supernatant liquid, add 8～12 ml sterile waters by 1 milliliter again, mix rear standingly more than 20 minutes, continue successively dilution, obtaining content is 10
-3～10
-6diluent, insert in beef-protein medium, temperature be under 39 ℃ dark cultivate 24 hours after, the single bacterium colony of picking is streak culture step by step, until form single bacterium colony.Described streak culture step by step: as impure bacterium colony to be chosen to single bacterium colony streak culture to single bacterium colony, for purifying bacterium colony not, continue to choose bacterium streak culture, until become single bacterium colony.
Actinomycetic separation and cultivation: the asafoetide explant after the cancellation poison, put into the mortar grinding of having sterilized and be pulpous state, by every 1 gram jelly, add 8～12 ml sterile waters, mix rear standingly more than 20 minutes, then therefrom draw supernatant liquid, add 8～12 ml sterile waters by 1 milliliter again, mix rear standingly more than 20 minutes, continue successively dilution, obtaining content is 10
-3～10
-5diluent, insert in the Gause I substratum dark the cultivation after 15 days, a small amount of mycelia in picking edge purifying is step by step cultivated, until form single bacterium colony.Described purifying step by step is: impure bacterium colony picking edge mycelia is placed in to the culture dish purifying and is cultured to single bacterium colony, for unpurified bacterium colony, continue picking edge purifying until become single bacterium colony.
embodiment 6:get the root of fresh Ferula sinkiangensisK.M.Shen, Ferula fukanensis K.M.Shen, sheep food asafoetide as explant, be cut into the segment of 2cm, through the alcohol disinfecting of concentration 70% 1 minute, then through the clorox sterilization of concentration 3% 5 minutes, sterilized water washing afterwards 4 times, draining the water is placed in 3 ℃ of environment and saves backup.
The separation of fungi and cultivation: by the explant of above-mentioned sterilization, be cut into the 0.3cm fritter and be inoculated in the wheat bran substratum, 5 of each culture dish inoculations; At 26 ℃ of temperature, secretly cultivate 6 days, after mycelia grows, picking mycelia tip, be placed in PDA substratum purifying step by step, until form single bacterium colony.Described purifying step by step is: impure bacterium colony picking edge mycelia is placed in to the culture dish purifying and is cultured to single bacterium colony, for unpurified bacterium colony, continue picking edge purifying until become single bacterium colony.
The separation of bacterium and cultivation: the asafoetide explant after the cancellation poison, put into the mortar grinding of having sterilized and be pulpous state, by every 1 gram jelly, add 8～12 ml sterile waters, mix rear standingly more than 20 minutes, then therefrom draw supernatant liquid, add 8～12 ml sterile waters by 1 milliliter again, mix rear standingly more than 20 minutes, continue successively dilution, obtaining content is 10
-3～10
-6diluent, insert in beef-protein medium, temperature be under 36～39 ℃ dark cultivate 24～48 hours after, the single bacterium colony of picking is streak culture step by step, until form single bacterium colony.Described streak culture step by step: as impure bacterium colony to be chosen to single bacterium colony streak culture to single bacterium colony, for purifying bacterium colony not, continue to choose bacterium streak culture, until become single bacterium colony.
Actinomycetic separation and cultivation: the asafoetide explant after the cancellation poison, put into the mortar grinding of having sterilized and be pulpous state, by every 1 gram jelly, add 8～12 ml sterile waters, mix rear standingly more than 20 minutes, then therefrom draw supernatant liquid, add 8～12 ml sterile waters by 1 milliliter again, mix rear standingly more than 20 minutes, continue successively dilution, obtaining content is 10
-2～10
-3diluent, insert in the Gause I substratum dark the cultivation after 14～21 days, a small amount of mycelia in picking edge purifying is step by step cultivated, until form single bacterium colony.Described purifying step by step is: impure bacterium colony picking edge mycelia is placed in to the culture dish purifying and is cultured to single bacterium colony, for unpurified bacterium colony, continue picking edge purifying until become single bacterium colony.
embodiment 7:get the root of fresh Ferula sinkiangensisK.M.Shen, Ferula fukanensis K.M.Shen, sheep food asafoetide as explant, be cut into the segment of 1.5cm, through the alcohol disinfecting of concentration 85% 1.5 minutes, then through the clorox sterilization of concentration 4% 5 minutes, sterilized water washing afterwards 5 times, drain to saving backup without dripping to be placed in 4 ℃ of environment.
The separation of fungi and cultivation: by the explant of above-mentioned sterilization, be cut into the 0.5cm fritter and be inoculated in the wheat bran substratum, 5 of each culture dish inoculations; At 24 ℃ of temperature, secretly cultivate 6 days, after mycelia grows, picking mycelia tip, be placed in PDA substratum purifying step by step, until form single bacterium colony.Described purifying step by step is: impure bacterium colony picking edge mycelia is placed in to the culture dish purifying and is cultured to single bacterium colony, for unpurified bacterium colony, continue picking edge purifying until become single bacterium colony.
The separation of bacterium and cultivation: the asafoetide explant after the cancellation poison, homogenate, then being diluted to content with sterilized water by weight is 10
-3～10
-6diluent, insert in beef-protein medium, temperature be under 37 ℃ dark cultivate 24 hours after, the single bacterium colony of picking is streak culture step by step, until form single bacterium colony.Described streak culture step by step: as impure bacterium colony to be chosen to single bacterium colony streak culture to single bacterium colony, for purifying bacterium colony not, continue to choose bacterium streak culture, until become single bacterium colony.
Actinomycetic separation and cultivation: the asafoetide explant after the cancellation poison, homogenate, then being diluted to content with sterilized water by weight is 10
-4～10
-5diluent, insert in the Gause I substratum dark the cultivation after 20 days, a small amount of mycelia in picking edge purifying is step by step cultivated, until form single bacterium colony.Described purifying step by step is: impure bacterium colony picking edge mycelia is placed in to the culture dish purifying and is cultured to single bacterium colony, for unpurified bacterium colony, continue picking edge purifying until become single bacterium colony.
embodiment 8:get the root of fresh Ferula sinkiangensisK.M.Shen, Ferula fukanensis K.M.Shen, sheep food asafoetide as explant, be cut into the segment of 1cm, through the alcohol disinfecting of concentration 70% 1 minute, then through the clorox sterilization of concentration 3% 5 minutes, sterilized water washing afterwards 4 times, draining the water is placed in 4 ℃ of environment and saves backup.
The separation of fungi and cultivation: by the explant of above-mentioned sterilization, be cut into the 0.5cm fritter and be inoculated in rose bengal medium, 5 of each culture dish inoculations; At 25 ℃ of temperature, secretly cultivate 5 days, after mycelia grows, picking mycelia tip, be placed in PDA substratum purifying step by step, until form single bacterium colony.Described purifying step by step is: impure bacterium colony picking edge mycelia is placed in to the culture dish purifying and is cultured to single bacterium colony, for unpurified bacterium colony, continue picking edge purifying until become single bacterium colony.
The separation of bacterium and cultivation: the asafoetide explant after the cancellation poison, homogenate, then being diluted to content with sterilized water by weight is 10
-3～10
-6diluent, insert in beef-protein medium, temperature be under 37 ℃ dark cultivate 48 hours after, the single bacterium colony of picking is streak culture step by step, until form single bacterium colony.Described streak culture step by step: as impure bacterium colony to be chosen to single bacterium colony streak culture to single bacterium colony, for purifying bacterium colony not, continue to choose bacterium streak culture, until become single bacterium colony.
Actinomycetic separation and cultivation: the asafoetide explant after the cancellation poison, homogenate, then being diluted to content with sterilized water by weight is 10
-2～10
-3diluent, insert in the Gause I substratum dark the cultivation after 18 days, a small amount of mycelia in picking edge purifying is step by step cultivated, until form single bacterium colony.Described purifying step by step is: impure bacterium colony picking edge mycelia is placed in to the culture dish purifying and is cultured to single bacterium colony, for unpurified bacterium colony, continue picking edge purifying until become single bacterium colony.
embodiment 9:get the root of fresh Ferula sinkiangensisK.M.Shen, Ferula fukanensis K.M.Shen, sheep food asafoetide as explant, be cut into the segment of 1.2cm, through alcohol disinfecting 1 clock of concentration 70%, then sterilize 6 minutes through the clorox of concentration 2%, sterilized water washing afterwards 3 times, draining the water is placed in 3 ℃ of environment and saves backup.
The separation of fungi and cultivation: by the explant of above-mentioned sterilization, be cut into 0.3～0.6cm fritter and be inoculated in rose bengal medium, 6 of each culture dish inoculations; At 27 ℃ of temperature, secretly cultivate 4 days, after mycelia grows, picking mycelia tip, be placed in PDA substratum purifying step by step, until form single bacterium colony.Described purifying step by step is: impure bacterium colony picking edge mycelia is placed in to the culture dish purifying and is cultured to single bacterium colony, for unpurified bacterium colony, continue picking edge purifying until become single bacterium colony.
The separation of bacterium and cultivation: the asafoetide explant after the cancellation poison, homogenate, then being diluted to content with sterilized water by weight is 10
-3～10
-6diluent, insert in beef-protein medium, temperature be under 36 ℃ dark cultivate 48 hours after, the single bacterium colony of picking is streak culture step by step, until form single bacterium colony.Described streak culture step by step: as impure bacterium colony to be chosen to single bacterium colony streak culture to single bacterium colony, for purifying bacterium colony not, continue to choose bacterium streak culture, until become single bacterium colony.
Actinomycetic separation and cultivation: the asafoetide explant after the cancellation poison, homogenate, then being diluted to content with sterilized water by weight is 10
-2～10
-5diluent, insert in the Gause I substratum dark the cultivation after 20 days, a small amount of mycelia in picking edge purifying is step by step cultivated, until form single bacterium colony.Described purifying step by step is: impure bacterium colony picking edge mycelia is placed in to the culture dish purifying and is cultured to single bacterium colony, for unpurified bacterium colony, continue picking edge purifying until become single bacterium colony.
embodiment 10:get the root of fresh Ferula sinkiangensisK.M.Shen, Ferula fukanensis K.M.Shen, sheep food asafoetide as explant, be cut into the segment of 0.8cm, through the alcohol disinfecting of concentration 75% 1.5 minutes, then through the clorox sterilization of concentration 3% 5 minutes, sterilized water washing afterwards 3 times, draining the water is placed in 3 ℃ of environment and saves backup.
The separation of fungi and cultivation: by the explant of above-mentioned sterilization, be cut into the 0.4cm fritter and be inoculated in the wheat bran substratum, 7 of each culture dish inoculations; At 25 ℃ of temperature, secretly cultivate 5 days, after mycelia grows, picking mycelia tip, be placed in PDA substratum purifying step by step, until form single bacterium colony.Described purifying step by step is: impure bacterium colony picking edge mycelia is placed in to the culture dish purifying and is cultured to single bacterium colony, for unpurified bacterium colony, continue picking edge purifying until become single bacterium colony.
The separation of bacterium and cultivation: the asafoetide explant after the cancellation poison, put into the mortar grinding of having sterilized and be pulpous state, by every 1 gram jelly, add 8～12 ml sterile waters, mix rear standingly more than 20 minutes, then therefrom draw supernatant liquid, add 8～12 ml sterile waters by 1 milliliter again, mix rear standingly more than 20 minutes, continue successively dilution, obtaining content is 10
-3～10
-6diluent, insert in beef-protein medium, temperature be under 38 ℃ dark cultivate 24 hours after, the single bacterium colony of picking is streak culture step by step, until form single bacterium colony.Described streak culture step by step: as impure bacterium colony to be chosen to single bacterium colony streak culture to single bacterium colony, for purifying bacterium colony not, continue to choose bacterium streak culture, until become single bacterium colony.
Actinomycetic separation and cultivation: the asafoetide explant after the cancellation poison, put into the mortar grinding of having sterilized and be pulpous state, by every 1 gram jelly, add 8～12 ml sterile waters, mix rear standingly more than 20 minutes, then therefrom draw supernatant liquid, add 8～12 ml sterile waters by 1 milliliter again, mix rear standingly more than 20 minutes, continue successively dilution, obtaining content is 10
-2～10
-5diluent, insert in the Gause I substratum dark the cultivation after 16 days, a small amount of mycelia in picking edge purifying is step by step cultivated, until form single bacterium colony.Described purifying step by step is: impure bacterium colony picking edge mycelia is placed in to the culture dish purifying and is cultured to single bacterium colony, for unpurified bacterium colony, continue picking edge purifying until become single bacterium colony.
Claims (9)
1. the expanding propagation method of a medicinal asafoetide endophyte is characterized in that mainly comprising following process:
Explant is made: get the root of fresh Ferula sinkiangensisK.M.Shen, Ferula fukanensis K.M.Shen, sheep food asafoetide as explant, be cut into the segment of 0.5～2cm, through the alcohol disinfecting of concentration 65～85% 0.5～1.5 minute, through the clorox of concentration 2～4%, sterilize 5～8 minutes again, sterilized water washing afterwards 3～5 times, draining the water is placed in 2～5 ℃ of environment and saves backup;
The separation of fungi and cultivation: by the explant of above-mentioned sterilization, be cut into 0.3～0.6cm fritter and be inoculated in wheat bran substratum or rose bengal medium substratum, 3～7 of each culture dish inoculations;
At 23～27 ℃ of temperature, secretly cultivate 4～8 days, after mycelia grows, picking mycelia tip, be placed in PDA substratum purifying step by step, until form single bacterium colony;
The separation of bacterium and cultivation: the asafoetide explant after the cancellation poison, homogenate, then being diluted to content with sterilized water by weight is 10
-3～10
-6diluent, insert in beef-protein medium, temperature be under 36～39 ℃ dark cultivate 24～48 hours after, the single bacterium colony of picking is streak culture step by step, until form single bacterium colony;
Actinomycetic separation and cultivation: the asafoetide explant after the cancellation poison, homogenate, then being diluted to content with sterilized water by weight is 10
-2～10
-5diluent, insert in the Gause I substratum dark the cultivation after 14～21 days, a small amount of mycelia in picking edge purifying is step by step cultivated, until form single bacterium colony.
2. the expanding propagation method of medicinal asafoetide endophyte according to claim 1, it is characterized in that: described purifying step by step is: impure bacterium colony picking edge mycelia is placed in to the culture dish purifying and is cultured to single bacterium colony, for unpurified bacterium colony, continue picking edge purifying until become single bacterium colony.
3. the expanding propagation method of medicinal asafoetide endophyte according to claim 1 and 2, it is characterized in that: described streak culture step by step: impure bacterium colony is chosen to single bacterium colony streak culture to single bacterium colony, for purifying bacterium colony not, continue to choose bacterium streak culture, until become single bacterium colony.
4. the expanding propagation method of medicinal asafoetide endophyte according to claim 1 and 2, it is characterized in that: described purifying step by step is: impure bacterium colony picking edge mycelia is placed in to the culture dish purifying and is cultured to single bacterium colony, for unpurified bacterium colony, continue picking edge purifying until become single bacterium colony.
5. the expanding propagation method of medicinal asafoetide endophyte according to claim 3, it is characterized in that: described purifying step by step is: impure bacterium colony picking edge mycelia is placed in to the culture dish purifying and is cultured to single bacterium colony, for unpurified bacterium colony, continue picking edge purifying until become single bacterium colony.
6. the expanding propagation method of medicinal asafoetide endophyte according to claim 1 and 2, it is characterized in that: described bacterium or actinomycetic diluent are prepared according to the following steps: the asafoetide explant after the cancellation poison, put into the mortar grinding of having sterilized and be pulpous state, by every 1 gram jelly, add 8～12 ml sterile waters, mix rear standing more than 20 minutes, then therefrom draw supernatant liquid, add 8～12 ml sterile waters by 1 milliliter again, mix rear standing more than 20 minutes, continue successively dilution, obtain required diluent.
7. the expanding propagation method of medicinal asafoetide endophyte according to claim 3, it is characterized in that: described bacterium or actinomycetic diluent are prepared according to the following steps: the asafoetide explant after the cancellation poison, put into the mortar grinding of having sterilized and be pulpous state, by every 1 gram jelly, add 8～12 ml sterile waters, mix rear standing more than 20 minutes, then therefrom draw supernatant liquid, add 8～12 ml sterile waters by 1 milliliter again, mix rear standing more than 20 minutes, continue successively dilution, obtain required diluent.
8. the expanding propagation method of medicinal asafoetide endophyte according to claim 4, it is characterized in that: described bacterium or actinomycetic diluent are prepared according to the following steps: the asafoetide explant after the cancellation poison, put into the mortar grinding of having sterilized and be pulpous state, by every 1 gram jelly, add 8～12 ml sterile waters, mix rear standing more than 20 minutes, then therefrom draw supernatant liquid, add 8～12 ml sterile waters by 1 milliliter again, mix rear standing more than 20 minutes, continue successively dilution, obtain required diluent.
9. the expanding propagation method of medicinal asafoetide endophyte according to claim 5, it is characterized in that: described bacterium or actinomycetic diluent are prepared according to the following steps: the asafoetide explant after the cancellation poison, put into the mortar grinding of having sterilized and be pulpous state, by every 1 gram jelly, add 8～12 ml sterile waters, mix rear standing more than 20 minutes, then therefrom draw supernatant liquid, add 8～12 ml sterile waters by 1 milliliter again, mix rear standing more than 20 minutes, continue successively dilution, obtain required diluent.
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