CN115918537B - Centella tissue culture rapid propagation method - Google Patents
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- CN115918537B CN115918537B CN202211595466.1A CN202211595466A CN115918537B CN 115918537 B CN115918537 B CN 115918537B CN 202211595466 A CN202211595466 A CN 202211595466A CN 115918537 B CN115918537 B CN 115918537B
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Abstract
The invention discloses a centella tissue culture rapid propagation method, which comprises the following steps: 1) Taking the stem section of centella asiatica with axillary buds, and sterilizing to obtain an aseptic centella asiatica explant; 2) Culturing sterile explants of centella asiatica by using an induction culture medium, and obtaining first-generation seedlings of centella asiatica after the explants germinate; 3) Culturing centella asiatica primary seedlings by using a proliferation culture medium to obtain primary seedling cluster buds; 4) Culturing primary seedling cluster buds by using a seedling strengthening culture medium to obtain centella seedling strengthening seedlings; 5) Culturing strong seedlings of centella asiatica by using a rooting medium until the root length is 2-4cm, and obtaining the seedlings of centella asiatica with roots; 6) And (3) hardening the centella asiatica with root seedlings, and transplanting the centella asiatica with root seedlings into a matrix for culturing. The invention can rapidly reproduce a large number of excellent centella seedlings suitable for transplanting, and meets the market demand.
Description
Technical Field
The invention relates to the technical field of tissue culture. More particularly, the invention relates to a centella tissue culture rapid propagation method.
Background
The Chinese medicine centella Centella asiatica (L.) Urb is dry whole herb of centella asiatica of Umbelliferae, which is similar to water chestnut or half copper coin, also called water chestnut, glechoma hederacea, herba Centellae, herba Potentillae Discoloris, etc. Is a traditional Chinese medicine in China, has a long history, is recorded in Shennong Ben Cao Jing at the earliest, is listed as a Chinese product, and has a harvest in Chinese pharmacopoeia. It is pungent, bitter and cold in nature, has the effects of clearing heat and promoting diuresis, and removing toxic substances and relieving swelling, and can be used for treating jaundice due to damp-heat, heatstroke diarrhea, stranguria with stone, carbuncle, swelling and sore due to blood stranguria, traumatic injury, etc. Centella (Centella) plants exist in about 20 or more regions around the world, are distributed in tropical and subtropical regions of the northern and southern hemispheres, are mainly produced in regions such as south Africa, india and Styraka, are also distributed in Japan, but only 1 variety of Centella is stored in China, and are largely distributed in regions such as east China, south China and southwest. Centella asiatica grows on wet and cloudy lands, field edges and ditch edges with the altitude of 200-1990 m, is loving sunlight and a relatively moist environment, and is strong in nature.
The modern clinical research results show that the centella asiatica has the functions of effectively easing pain, resisting tumor, resisting microorganisms, regulating immunity and the like, and can be collected in four seasons, fresh or dried in the sun. The market demand has increased in recent years. However, the existing centella raw materials mainly depend on wild resources, the wild centella is scattered, plants are small, growth is slow, collection is difficult, and meanwhile, the centella resources are reduced due to the use of pesticide such as folk herbicides, so that the centella price rises year by year, the wild centella resources face endangerment, and therefore introduction cultivation, artificial breeding and the like become a new way for effectively utilizing the centella resources. In recent years, centella asiatica is widely researched and clinically applied, but researchers have many researches on chemical components and pharmacological actions, few researches on propagation and researches on tissue culture propagation by using explants thereof and only few reports on polyploidy, wherein He Gong et al disclose an in vitro culture and rapid propagation method of centella asiatica to study, and the researches show that when the explants of the centella asiatica with the stem segments are induced and cultured, the induction rate of adventitious buds can reach 87.6 percent of the highest, and when 0.5mg/L BA is used, the induction rate of adventitious buds is only 26.8 percent, and in addition, the test-tube seedlings of centella asiatica with good plant growth and more root systems are transplanted, the survival rate of the transplanted centella asiatica can reach 90 percent, and obviously, the success rate of the survival rate of the finally obtained transplanted centella asiatica from the induced differentiation bud emergence to the final culture of the tissue culture of centella asiatica disclosed in the technology is lower than 80 percent. In addition, the applicant finds that centella is sensitive to TDZ in the experimental process, and the TDZ can influence the growth vigor of centella primary seedlings to a certain extent during induction culture, even the shrinkage of centella leaves can be caused, and the survival rate of transplanting after rooting of centella tissue culture seedlings is influenced to a certain extent.
Therefore, a more efficient centella tissue culture rapid propagation system is established, the success rate of centella tissue culture rapid propagation can be improved, a large amount of centella aseptic seedlings can be obtained efficiently, and the centella tissue culture rapid propagation system has a wide market application prospect.
Disclosure of Invention
The invention aims to provide a method for carrying out centella tissue culture and rapid propagation by utilizing centella with axillary bud stem segments, which can rapidly propagate a large number of excellent centella seedlings suitable for transplanting and meet market demands.
In order to achieve the aim and other advantages of the present invention, there is provided a rapid propagation method of centella asiatica by tissue culture, comprising the steps of:
1) Taking the stem section of centella asiatica with axillary buds, and sterilizing to obtain an aseptic centella asiatica explant;
2) Culturing sterile explant of centella asiatica by using an induction culture medium, and obtaining first-generation seedlings of centella asiatica after the explant germinates, wherein the induction culture medium contains 1.5-2.5mg/L of 6-benzylaminoadenine and 0.1-1.0mg/L of kinetin;
3) Culturing herba Centellae primary seedlings by using proliferation medium to obtain primary seedling cluster buds, wherein the proliferation medium contains 0.05-0.35mg/L indoleacetic acid, 0-0.5 mg/L6-benzylaminoadenine, and 0.1-1.0mg/L kinetin;
4) Culturing primary seedling cluster buds by using a seedling strengthening culture medium to obtain centella asiatica seedling strengthening culture medium, wherein the seedling strengthening culture medium contains 0.1-0.5mg/L of 6-benzylaminoadenine, 0.1-0.5mg/L of indoleacetic acid and 0.2-0.8g/L of activated carbon;
5) Culturing strong centella asiatica seedlings by using a rooting culture medium until the roots grow to 2-4cm, and obtaining centella asiatica seedlings with roots, wherein the rooting culture medium contains 0.1-0.5mg/L of indoleacetic acid, 0-0.5mg/L of naphthylacetic acid and 0.5-1.0g/L of activated carbon;
6) And (3) hardening the centella asiatica with root seedlings, and transplanting the centella asiatica with root seedlings into a matrix for culturing.
Preferably, in the centella asiatica tissue culture rapid propagation method, an MS culture medium is adopted as an induction culture medium, a proliferation culture medium and a strong seedling culture medium, a 1/2MS culture medium is adopted as a rooting culture medium, and the induction culture medium, the proliferation culture medium, the strong seedling culture medium and the rooting culture medium all contain 5.0g/L of agar and 30g/L of sucrose, and have pH values of 5.8.
Preferably, in the centella asiatica tissue culture rapid propagation method, in the step 2), the culture temperature is 24-26 ℃, the illumination intensity is 1500lux, the illumination time is 10-12 hours/day, and the culture is 15-30 days; in the step 3), the culture temperature is 24-26 ℃, the illumination intensity is 2000lux, the illumination time is 10-12 hours/day, and the culture is carried out for 30 days; in the step 4), the culture temperature is 24-26 ℃, the illumination intensity is 2000lux, the illumination time is 10-12 hours/day, and the culture is 10-21 days; in the step 5), the culture temperature is 24-26 ℃, the illumination intensity is 2000lux, the illumination time is 10-12 hours/day, and the culture is carried out for 21 days.
Preferably, in the centella asiatica tissue culture rapid propagation method, the induction medium contains 2mg/L of 6-benzylaminoadenine and 0.5mg/L of kinetin; the proliferation medium contains 0.2mg/L indoleacetic acid, 0.25 mg/L6-benzylaminoadenine and 0.5mg/L kinetin; the strong seedling culture medium contains 0.3mg/L of 6-benzylaminoadenine, 0.3mg/L of indoleacetic acid and 0.5g/L of activated carbon; the rooting culture medium contains 0.3mg/L of indoleacetic acid, 0.25mg/L of naphthylacetic acid and 0.75g/L of activated carbon.
Preferably, in the centella asiatica tissue culture rapid propagation method, in the step 2), the induction culture time is 15 days, 0.1mg/L of lingfebrile extract and 2mg/L of nano zinc oxide are also contained in the induction culture, the illumination culture is carried out under the alternate environment of white light and red and blue light, the white light is firstly illuminated for 9 hours/day, then the red and blue light is illuminated for 3 hours/day, finally the dark culture is carried out for 12 hours/day, and the cycle is carried out in sequence, wherein the ratio of red light to blue light is 2:1.
Preferably, in the centella asiatica tissue culture rapid propagation method, in the step 4), the strong seedling culture time is 10 days, the strong seedling culture medium also contains 0.2g/L of hydrolyzed casein and 20 mu mol/L of methyl jasmonate, the illumination is carried out under the alternate environment of white light and red and blue light during illumination culture, the white light is firstly illuminated for 8 hours/day, then the red and blue light is illuminated for 4 hours/day, finally the dark culture is carried out for 12 hours/day, and the culture is sequentially circulated, wherein the ratio of red light to blue light is 2:3.
Preferably, the centella asiatica tissue culture rapid propagation method, in the step 1), the disinfection process is specifically as follows: sequentially soaking the stems with axillary buds of centella asiatica with aqueous solution of liquid detergent with mass concentration of 1% for 5-10min, washing with tap water for 15-30min, washing with sterile water once, soaking with alcohol with volume concentration of 75% for 30-50s, sterilizing with 150ml of 0.1% mercuric chloride added with 2-3 drops of Tween-80 for 6-8min, washing with sterile water for 3-5 times, and spreading on sterilized filter paper to remove surface moisture.
Preferably, in the centella tissue culture rapid propagation method, the transplanted substrate consists of seedling raising soil, sheep manure and vermiculite according to the mass ratio of 5:1:1.
Preferably, the centella asiatica tissue culture rapid propagation method comprises the following specific processes of: removing centella asiatica with root seedling together with culture flask, transferring into greenhouse, standing at 25-27deg.C for 7 days, opening bottle cap, adding tap water, and hardening off seedling for 3-4 days.
Preferably, in the centella asiatica tissue culture rapid propagation method, a plastic film is used for moisturizing in the first 7-10 days after transplanting, a layer of newspaper is covered on the plastic film, the shading rate is 60% -70%, the air humidity is 85% -90%, and spraying is carried out for 1 time every day.
The invention at least comprises the following beneficial effects:
(1) The centella tissue culture seedling culture process is easy to manage by discarding the sensitive TDZ hormone of centella, so that the centella tissue culture seedling with good growth vigor and good quality is obtained;
(2) The transplanting survival rate of centella asiatica with root seedlings is improved by carrying out strong seedling culture before rooting;
(3) The induction culture medium is added with the lingfa with proper concentration and the nano zinc oxide, and the induced culture is performed under the specific illumination condition, so that the induction rate of the induced culture is further improved to 100% under the condition of shortening the induction culture time, and the primary seedlings with better growth vigor are obtained, thereby providing a good foundation for the subsequent culture;
(4) The hydrolyzed casein and methyl jasmonate with proper concentrations are added into the strong seedling culture medium, and the culture is carried out under specific illumination, so that the growth vigor of primary seedling cluster buds is further enhanced under the condition of shortening the strong seedling culture time, and the transplanting survival rate of the centella asiatica with roots is improved to 100%.
Additional advantages, objects, and features of the invention will be set forth in part in the description which follows and in part will become apparent to those having ordinary skill in the art upon examination of the following or may be learned from practice of the invention.
Detailed Description
The present invention is described in further detail below with reference to examples to enable those skilled in the art to practice the same by referring to the description.
The experimental methods described in the examples below are conventional methods unless otherwise indicated, and the reagents and materials described herein are commercially available.
A centella asiatica tissue culture rapid propagation method comprises the following steps:
1) Taking the stem section of centella asiatica with axillary buds, and sterilizing to obtain an aseptic centella asiatica explant;
2) Culturing sterile explant of centella asiatica by using an induction culture medium, and obtaining first-generation seedlings of centella asiatica after the explant germinates, wherein the induction culture medium contains 1.5-2.5mg/L of 6-benzylaminoadenine and 0.1-1.0mg/L of kinetin;
3) Culturing herba Centellae primary seedlings by using proliferation medium to obtain primary seedling cluster buds, wherein the proliferation medium contains 0.05-0.35mg/L indoleacetic acid, 0-0.5 mg/L6-benzylaminoadenine, and 0.1-1.0mg/L kinetin;
4) Culturing primary seedling cluster buds by using a seedling strengthening culture medium to obtain centella asiatica seedling strengthening culture medium, wherein the seedling strengthening culture medium contains 0.1-0.5mg/L of 6-benzylaminoadenine, 0.1-0.5mg/L of indoleacetic acid and 0.2-0.8g/L of activated carbon;
5) Culturing strong centella asiatica seedlings by using a rooting culture medium until the roots grow to 2-4cm, and obtaining centella asiatica seedlings with roots, wherein the rooting culture medium contains 0.1-0.5mg/L of indoleacetic acid, 0-0.5mg/L of naphthylacetic acid and 0.5-1.0g/L of activated carbon;
6) And (3) hardening the centella asiatica with root seedlings, and transplanting the centella asiatica with root seedlings into a matrix for culturing.
The basal culture used in the induction culture medium, the proliferation culture medium, the strong seedling culture medium and the rooting culture medium in the technical scheme can be MS culture medium or MT culture medium. By carrying out strong seedling culture before rooting, the transplanting survival rate of centella asiatica with root seedlings can be improved; in addition, the technical proposal eliminates the use of the sensitive TDZ hormone of centella asiatica, so that the culture process of centella asiatica tissue culture seedlings is easy to manage, and centella asiatica tissue culture seedlings with good growth vigor and good quality are obtained.
More preferably, the centella tissue culture rapid propagation method adopts an MS culture medium as an induction culture medium, a proliferation culture medium and a strong seedling culture medium, adopts a 1/2MS culture medium as a rooting culture medium, and comprises 5.0g/L of agar and 30g/L of sucrose as the induction culture medium, the proliferation culture medium, the strong seedling culture medium and the rooting culture medium, and has a pH value of 5.8. After the culture medium used in the invention is prepared, the culture medium is bottled and filled, and then high-pressure sterilization is carried out, wherein the sterilization temperature is 121 ℃, and the sterilization time is 20min.
More preferably, in the centella asiatica tissue culture rapid propagation method, in the step 2), the culture temperature is 24-26 ℃, the illumination intensity is 1500lux, the illumination time is 10-12 hours/day, and the culture is 15-30 days; in the step 3), the culture temperature is 24-26 ℃, the illumination intensity is 2000lux, the illumination time is 10-12 hours/day, and the culture is carried out for 30 days; in the step 4), the culture temperature is 24-26 ℃, the illumination intensity is 2000lux, the illumination time is 10-12 hours/day, and the culture is 10-21 days; in the step 5), the culture temperature is 24-26 ℃, the illumination intensity is 2000lux, the illumination time is 10-12 hours/day, and the culture is carried out for 21 days. In the technical scheme, the illumination treatment is white light.
More preferably, the centella asiatica tissue culture rapid propagation method comprises the step of inducing 2mg/L of 6-benzylaminoadenine and 0.5mg/L of kinetin in a culture medium; the proliferation medium contains 0.2mg/L indoleacetic acid, 0.25 mg/L6-benzylaminoadenine and 0.5mg/L kinetin; the strong seedling culture medium contains 0.3mg/L of 6-benzylaminoadenine, 0.3mg/L of indoleacetic acid and 0.5g/L of activated carbon; the rooting culture medium contains 0.3mg/L of indoleacetic acid, 0.25mg/L of naphthylacetic acid and 0.75g/L of activated carbon.
More preferably, in the centella asiatica tissue culture rapid propagation method, in the step 2), the induction culture time is 15 days, 0.1mg/L of lingfebrile extract and 2mg/L of nano zinc oxide are also contained in the induction culture, the illumination culture is carried out under the alternate environment of white light and red and blue light, the white light is firstly illuminated for 9 hours/day, then the red and blue light is illuminated for 3 hours/day, finally the dark culture is carried out for 12 hours/day, and the cycle is carried out in sequence, wherein the ratio of red light to blue light is 2:1. According to the technical scheme, the ling fa and the nano zinc oxide with proper concentrations are added into the induction culture medium, and the induction culture is performed under specific illumination conditions, so that the induction rate of the induction culture is further improved under the condition of shortening the induction culture time, and the primary seedlings with better growth vigor are obtained, so that a good foundation is provided for subsequent culture.
More preferably, in the centella asiatica tissue culture rapid propagation method, in the step 4), the strong seedling culture time is 10 days, the strong seedling culture medium also contains 0.2g/L of hydrolyzed casein and 20 mu mol/L of methyl jasmonate, the illumination is carried out under the alternate environment of white light and red and blue light during illumination culture, the white light is firstly illuminated for 8 hours/day, then the red and blue light is illuminated for 4 hours/day, finally the dark culture is carried out for 12 hours/day, and the culture is sequentially circulated, wherein the ratio of red light to blue light is 2:3. According to the technical scheme, hydrolyzed casein and methyl jasmonate with proper concentrations are added into the strong seedling culture medium, and the strong seedling culture medium is cultured under specific illumination, so that the growth vigor of primary seedling cluster buds is further enhanced under the condition of shortening the strong seedling culture time, and the transplanting survival rate of centella asiatica with roots is improved to 100%.
More preferably, the centella asiatica tissue culture rapid propagation method, in the step 1), the disinfection process is specifically as follows: sequentially soaking the stems with axillary buds of centella asiatica with aqueous solution of liquid detergent with mass concentration of 1% for 5-10min, washing with tap water for 15-30min, washing with sterile water once, soaking with alcohol with volume concentration of 75% for 30-50s, sterilizing with 150ml of 0.1% mercuric chloride added with 2-3 drops of Tween-80 for 6-8min, washing with sterile water for 3-5 times, and spreading on sterilized filter paper to remove surface moisture.
More preferably, in the centella tissue culture rapid propagation method, the transplanted substrate consists of seedling raising soil, sheep manure and vermiculite according to the mass ratio of 5:1:1.
More preferably, the centella asiatica tissue culture rapid propagation method comprises the following specific processes of seedling hardening in the step 6): removing centella asiatica with root seedling together with culture flask, transferring into greenhouse, standing at 25-27deg.C for 7 days, opening bottle cap, adding tap water, and hardening off seedling for 3-4 days.
More preferably, the centella asiatica tissue culture rapid propagation method uses plastic films for moisturizing in the first 7-10 days after transplanting, a layer of newspaper is covered on the plastic films, the shading rate is 60% -70%, the air humidity is 85% -90%, and spraying is carried out for 1 time every day.
The technical scheme of the invention can also comprise the following technical details so as to better realize the technical effects:
example 1:
a centella asiatica tissue culture rapid propagation method comprises the following steps:
1) Taking the stem section of centella asiatica with axillary buds, and sterilizing to obtain an aseptic centella asiatica explant;
2) Culturing sterile explant of centella asiatica by using an induction culture medium, and obtaining first-generation seedlings of centella asiatica after the explant germinates, wherein the induction culture medium contains 1.5mg/L of 6-benzylaminoadenine and 0.1mg/L of kinetin;
3) Culturing centella asiatica primary seedlings by using a proliferation culture medium to obtain primary seedling cluster buds, wherein the proliferation culture medium contains 0.05mg/L of indoleacetic acid and 0.1mg/L of kinetin;
4) Culturing primary seedling cluster buds by using a seedling strengthening culture medium to obtain centella asiatica seedling strengthening culture medium, wherein the seedling strengthening culture medium contains 0.1mg/L of 6-benzylaminoadenine, 0.1mg/L of indoleacetic acid and 0.2g/L of activated carbon;
5) Culturing strong seedlings of centella asiatica until the root length is 2-4cm by using a rooting medium to obtain seedlings of centella asiatica with roots, wherein the rooting medium contains 0.1mg/L of indoleacetic acid and 0.5g/L of activated carbon;
6) And (3) hardening the centella asiatica with root seedlings, and transplanting the centella asiatica with root seedlings into a matrix for culturing.
Wherein, the inducing culture medium, the proliferation culture medium and the seedling strengthening culture medium are all MS culture medium, the rooting culture medium is 1/2MS culture medium, and the inducing culture medium, the proliferation culture medium, the seedling strengthening culture medium and the rooting culture medium all contain 5.0g/L agar and 30g/L sucrose, and the pH value is 5.8.
Wherein, in the step 2), the culture temperature is 24-26 ℃, the illumination intensity is 1500lux, the illumination time is 10 hours/day, and the culture is carried out for 30 days; in the step 3), the culture temperature is 24-26 ℃, the illumination intensity is 2000lux, the illumination time is 10 hours/day, and the culture is carried out for 30 days; in the step 4), the culture temperature is 24-26 ℃, the illumination intensity is 2000lux, the illumination time is 10 hours/day, and the culture is carried out for 21 days; in the step 5), the culture temperature is 24-26 ℃, the illumination intensity is 2000lux, the illumination time is 10 hours/day, and the culture is carried out for 21 days.
Wherein, the disinfection process in the step 1) is specifically as follows: sequentially soaking the stems with axillary buds of centella asiatica with aqueous solution of liquid detergent with mass concentration of 1% for 5min, washing with tap water for 15min, washing with sterile water once, soaking with 75% alcohol for 30s, sterilizing with 150ml of 0.1% mercuric chloride added with 2 drops of tween-80 for 6min, washing with sterile water for 3 times, and spreading on sterilized filter paper to remove surface water.
The transplanted substrate consists of seedling raising soil, sheep manure and vermiculite according to the mass ratio of 5:1:1.
Wherein, the concrete process of hardening off in step 6) is: removing herba Centellae with root and culture bottle, transferring into greenhouse, standing at 25-27deg.C for 7 days, opening bottle cap, adding tap water, and hardening off for 3 days.
Wherein, the plastic film is used for moisturizing in the first 7-10 days after transplanting, a layer of newspaper is covered on the plastic film, the shading rate is 60-70%, the air humidity is 85-90%, and the plastic film is sprayed for 1 time every day.
Example 2:
a centella asiatica tissue culture rapid propagation method comprises the following steps:
1) Taking the stem section of centella asiatica with axillary buds, and sterilizing to obtain an aseptic centella asiatica explant;
2) Culturing sterile explant of centella asiatica by using an induction culture medium, and obtaining first-generation seedlings of centella asiatica after the explant germinates, wherein the induction culture medium contains 2.5mg/L of 6-benzylaminoadenine and 1.0mg/L of kinetin;
3) Culturing centella asiatica primary seedlings by using a proliferation culture medium to obtain primary seedling cluster buds, wherein the proliferation culture medium contains 0.35mg/L of indoleacetic acid, 0.5mg/L of 6-benzylaminoadenine and 1.0mg/L of kinetin;
4) Culturing primary seedling cluster buds by using a seedling strengthening culture medium to obtain centella asiatica seedling strengthening culture medium, wherein the seedling strengthening culture medium contains 0.5mg/L of 6-benzylaminoadenine, 0.5mg/L of indoleacetic acid and 0.8g/L of activated carbon;
5) Culturing strong centella seedlings by using a rooting medium until the roots grow to 2-4cm to obtain centella seedlings with roots, wherein the rooting medium contains 0.5mg/L of indoleacetic acid, 0.5mg/L of naphthylacetic acid and 1.0g/L of activated carbon;
6) And (3) hardening the centella asiatica with root seedlings, and transplanting the centella asiatica with root seedlings into a matrix for culturing.
Wherein, the inducing culture medium, the proliferation culture medium and the seedling strengthening culture medium are all MS culture medium, the rooting culture medium is 1/2MS culture medium, and the inducing culture medium, the proliferation culture medium, the seedling strengthening culture medium and the rooting culture medium all contain 5.0g/L agar and 30g/L sucrose, and the pH value is 5.8.
Wherein, in the step 2), the culture temperature is 24-26 ℃, the illumination intensity is 1500lux, the illumination time is 12 hours/day, and the culture is carried out for 30 days; in the step 3), the culture temperature is 24-26 ℃, the illumination intensity is 2000lux, the illumination time is 12 hours/day, and the culture is carried out for 30 days; in the step 4), the culture temperature is 24-26 ℃, the illumination intensity is 2000lux, the illumination time is 12 hours/day, and the culture is carried out for 21 days; in the step 5), the culture temperature is 24-26 ℃, the illumination intensity is 2000lux, the illumination time is 12 hours/day, and the culture is carried out for 21 days.
Wherein, the disinfection process in the step 1) is specifically as follows: sequentially soaking the stems with axillary buds of centella asiatica in a liquid detergent water solution with the mass concentration of 1% for 10min, washing with tap water for 30min, washing with sterile water once, soaking in alcohol with the volume concentration of 75% for 50s, sterilizing with 150ml of mercury chloride with the mass concentration of 0.1% added with 3 drops of tween-80 for 8min, washing with sterile water for 5 times, and spreading on sterilized filter paper to remove surface moisture.
The transplanted substrate consists of seedling raising soil, sheep manure and vermiculite according to the mass ratio of 5:1:1.
Wherein, the concrete process of hardening off in step 6) is: removing herba Centellae with root and culture bottle, transferring into greenhouse, standing at 25-27deg.C for 7 days, opening bottle cap, adding tap water, and hardening off for 4 days.
Wherein, the plastic film is used for moisturizing in the first 7-10 days after transplanting, a layer of newspaper is covered on the plastic film, the shading rate is 60-70%, the air humidity is 85-90%, and the plastic film is sprayed for 1 time every day.
Example 3:
a centella asiatica tissue culture rapid propagation method comprises the following steps:
1) Taking the stem section of centella asiatica with axillary buds, and sterilizing to obtain an aseptic centella asiatica explant;
2) Culturing sterile explant of centella asiatica by using an induction culture medium, and obtaining first-generation seedlings of centella asiatica after the explant germinates, wherein the induction culture medium contains 2mg/L of 6-benzylaminoadenine and 0.5mg/L of kinetin;
3) Culturing centella asiatica primary seedlings by using a proliferation culture medium to obtain primary seedling cluster buds, wherein the proliferation culture medium contains 0.2mg/L of indoleacetic acid, 0.25mg/L of 6-benzylaminoadenine and 0.5mg/L of kinetin;
4) Culturing primary seedling cluster buds by using a seedling strengthening culture medium to obtain centella asiatica seedling strengthening culture medium, wherein the seedling strengthening culture medium contains 0.3mg/L of 6-benzylaminoadenine, 0.3mg/L of indoleacetic acid and 0.5g/L of activated carbon;
5) Culturing strong centella asiatica seedlings by using a rooting medium until the roots grow to 2-4cm to obtain centella asiatica seedlings with roots, wherein the rooting medium contains 0.3mg/L of indoleacetic acid, 0.25mg/L of naphthylacetic acid and 075g/L of activated carbon;
6) And (3) hardening the centella asiatica with root seedlings, and transplanting the centella asiatica with root seedlings into a matrix for culturing.
Wherein, the inducing culture medium, the proliferation culture medium and the seedling strengthening culture medium are all MS culture medium, the rooting culture medium is 1/2MS culture medium, and the inducing culture medium, the proliferation culture medium, the seedling strengthening culture medium and the rooting culture medium all contain 5.0g/L agar and 30g/L sucrose, and the pH value is 5.8.
Wherein, in the step 2), the culture temperature is 24-26 ℃, the illumination intensity is 1500lux, the illumination time is 12 hours/day, and the culture is carried out for 30 days; in the step 3), the culture temperature is 24-26 ℃, the illumination intensity is 2000lux, the illumination time is 12 hours/day, and the culture is carried out for 30 days; in the step 4), the culture temperature is 24-26 ℃, the illumination intensity is 2000lux, the illumination time is 12 hours/day, and the culture is carried out for 21 days; in the step 5), the culture temperature is 24-26 ℃, the illumination intensity is 2000lux, the illumination time is 12 hours/day, and the culture is carried out for 21 days.
Wherein, the disinfection process in the step 1) is specifically as follows: sequentially soaking the stems with axillary buds of centella asiatica in a liquid detergent water solution with the mass concentration of 1% for 8min, washing with tap water for 20min, washing with sterile water once, soaking with alcohol with the volume concentration of 75% for 40s, sterilizing with 150ml of mercury chloride with the mass concentration of 0.1% added with 2 drops of tween-80 for 7min, washing with sterile water for 4 times, and spreading on sterilized filter paper to remove surface moisture.
The transplanted substrate consists of seedling raising soil, sheep manure and vermiculite according to the mass ratio of 5:1:1.
Wherein, the concrete process of hardening off in step 6) is: removing herba Centellae with root and culture bottle, transferring into greenhouse, standing at 25-27deg.C for 7 days, opening bottle cap, adding tap water, and hardening off for 3 days.
Wherein, the plastic film is used for moisturizing in the first 7-10 days after transplanting, a layer of newspaper is covered on the plastic film, the shading rate is 60-70%, the air humidity is 85-90%, and the plastic film is sprayed for 1 time every day.
Example 4:
based on the embodiment 3, in the centella asiatica tissue culture rapid propagation method, in the step 2), the induction culture time is 15 days, 0.1mg/L of lingfebrile element and 2mg/L of nano zinc oxide are also contained in the induction culture, the illumination treatment is carried out under the alternate environment of white light and red and blue light during illumination culture, the white light is firstly illuminated for 9 hours/day, then the red and blue light is illuminated for 3 hours/day, finally the dark culture is carried out for 12 hours/day, and the steps are sequentially circulated, wherein the ratio of red light to blue light is 2:1. The culture temperature and the illumination intensity were the same as in example 3, and the rest was the same as in example 3.
Example 5:
based on the embodiment 3, in the centella asiatica tissue culture rapid propagation method, in the step 4), the strong seedling culture time is 10 days, the strong seedling culture medium also contains 0.2g/L of hydrolyzed casein and 20 mu mol/L of methyl jasmonate, the illumination culture is carried out under the alternate environment of white light and red and blue light, the illumination of the white light is carried out for 8 hours/day, the illumination of the red and blue light is carried out for 4 hours/day, the dark culture is carried out for 12 hours/day, and the circulation is carried out in sequence, wherein the ratio of the red light to the blue light is 2:3. The culture temperature and the illumination intensity were the same as in example 3, and the rest was the same as in example 3.
Example 6:
based on the embodiment 4, in the centella asiatica tissue culture rapid propagation method, in the step 4), the strong seedling culture time is 10 days, the strong seedling culture medium also contains 0.2g/L of hydrolyzed casein and 20 mu mol/L of methyl jasmonate, the illumination culture is carried out under the alternate environment of white light and red and blue light, the illumination of the white light is carried out for 8 hours/day, the illumination of the red and blue light is carried out for 4 hours/day, the dark culture is carried out for 12 hours/day, and the circulation is carried out in sequence, wherein the ratio of the red light to the blue light is 2:3. The culture temperature and the illumination intensity were the same as in example 4, and the rest was the same as in example 4.
Comparative example 1:
on the basis of example 3, the rooting culture was performed directly on primary cluster buds obtained by multiplication culture in the process of removing strong seedlings, and the rest was the same as in example 3.
Comparative example 2:
on the basis of example 3, the induction medium was replaced with: MS+0.5mg/L TDZ, the remainder being as in example 3.
Comparative example 3:
on the basis of example 4, 2mg/L of nano zinc oxide was removed from the induction medium, and the rest was the same as example 4.
Comparative example 4:
based on example 4, 0.1mg/L of lingfa was removed from the induction medium, with the remainder being the same as in example 4.
Comparative example 5:
on the basis of example 4, the conditions of the light culture (light source and light intensity) were the same as those of example 3, and the white light was irradiated for 12 hours/day, and the rest was the same as that of example 4.
Comparative example 6:
based on example 6, 20. Mu. Mol/L methyl jasmonate was removed from the medium for strong seedlings, and the rest was the same as in example 6.
Comparative example 7:
on the basis of example 6, 0.2g/L of hydrolyzed casein was removed from the strong seedling medium, with the remainder being the same as in example 6.
Comparative example 8:
on the basis of example 6, when strong seedlings are cultivated, the illumination cultivation conditions (light source and light intensity) are the same as those of example 3, and white light is illuminated for 12 hours/day, and the rest is the same as that of example 6.
In order to better highlight the beneficial effects of the centella asiatica tissue culture rapid propagation method of the invention, the inventor adopts centella asiatica with buds stem segments (from centella asiatica stem segments which grow robustly and have no plant diseases and insect pests, cut into small stem segments with buds of 1cm for use) obtained from the same source, and carries out tissue culture aiming at the technical schemes of examples 3-6 and comparative examples 1-8, wherein: during induction culture, 20 bottles of samples are used for each technical scheme, each bottle is inoculated with 1 explant, the induction culture process is observed, the induction effect is recorded, and the induction rate is calculated; cutting the obtained centella primary seedling with axillary bud into small segments of about 1cm, inoculating into proliferation culture medium for proliferation culture, treating 5 bottles in each technical scheme, inoculating 4 small segments into each bottle, observing and recording proliferation coefficient; dividing primary seedling cluster buds obtained by multiplication culture into about 2-3 cm single plants, inoculating strong seedling culture mediums to carry out strong seedling culture, treating 5 bottles of each culture medium, inoculating 4 seedlings in each bottle, changing rooting culture mediums after the strong seedling culture is completed, and observing and recording rooting conditions; and performing hardening and transplanting treatment on centella asiatica tissue culture seedlings obtained through rooting culture, and counting the transplanting survival rate after 30 days.
Stem induction = (number of budded explants/total number of explants) ×100%; proliferation coefficient= (total number of clump buds proliferated/total number of inoculated explants) ×100%; rooting rate= (number of plants with roots/number of plants inoculated) ×100%; transplanting survival rate= (number of surviving plants/number of transplanted plants) ×100%.
TABLE 1 influence of different protocols on the induction of stem segments
TABLE 2 influence of different protocols on proliferation, rooting and transplanting of tissue culture seedlings
As can be seen from table 1, compared with example 3, in example 4, the addition of the proper concentration of the lingfa and the nano zinc oxide in the induction culture combined with the specific red and blue light treatment can shorten the induction culture time, increase the induction rate of the induction culture to 100%, and obtain the primary seedlings with good growth vigor; in example 4, compared with comparative examples 3 to 5, the induction culture is deficient in 3 factors of the lingfa, the nano zinc oxide and the red and blue light, and in particular, as can be seen from comparative example 5 lacking red and blue light treatment, the lingfa and the nano zinc oxide can promote the induction acceleration of centella asiatica explants, but without combining with the red and blue light treatment, larger callus tissues are easy to appear on the primary seedling foundation, the browning speed of leaves is accelerated, and the growth vigor of the primary seedling is influenced.
As can be seen from Table 2, compared with example 4, the embodiment 6 adds hydrolyzed casein and methyl jasmonate during the cultivation of strong seedlings, and the cultivation under specific red and blue light can shorten the cultivation time of strong seedlings, and enhance the growth vigor of the cluster buds of the primary seedlings and the root seedlings of centella asiatica, thereby improving the transplanting survival rate of the root seedlings of centella asiatica to 100%. Compared with comparative examples 6-8, the addition of hydrolyzed casein, methyl jasmonate and 3 factors of red and blue light to the strong seedling culture medium is indispensable, especially hydrolyzed casein, which provides proper nutrients for the seedling replacing cluster buds and the centella asiatica with root seedlings, promotes the plants to be stronger and easy to transplant and survive.
As can be seen from example 3 and comparative example 2, the rapid propagation method of centella asiatica tissue culture of the present invention does not use TDZ hormone having a better induction effect on centella asiatica, but also obtains centella asiatica plants which are induced to propagate by using TDZ hormone, but have the same induction rate, transplanting survival rate and vigor.
Although embodiments of the present invention have been disclosed above, it is not limited to the use of the description and embodiments, it is well suited to various fields of use for the invention, and further modifications may be readily apparent to those skilled in the art, and accordingly, the invention is not limited to the particular details without departing from the general concepts defined in the claims and the equivalents thereof.
Claims (4)
1. The centella tissue culture rapid propagation method is characterized by comprising the following steps of:
1) Taking the stem section of centella asiatica with axillary buds, and sterilizing to obtain an aseptic centella asiatica explant;
2) Culturing an aseptic centella explant by using an induction culture medium, obtaining an initial centella seedling after the explant germinates, wherein the culture temperature is 24-26 ℃, the illumination intensity is 1500lux, the illumination time is 12 hours/day, the culture is 15 days, the illumination treatment is carried out under the alternate environment of white light and red blue light during illumination culture, the white light is firstly illuminated for 9 hours/day, then the red blue light is illuminated for 3 hours/day, and finally the dark culture is carried out for 12 hours/day, the cycle is sequentially carried out, wherein the ratio of red light to blue light is 2:1, the induction culture medium contains 2mg/L of 6-benzylaminoadenine, 0.5mg/L of kinetin, 0.1mg/L of lingfa and 2mg/L of nano zinc oxide;
3) Culturing centella asiatica primary seedlings by using a proliferation culture medium to obtain primary seedling cluster buds, wherein the culture temperature is 24-26 ℃, the illumination intensity is 2000lux, the illumination time is 10-12 hours/day, and the culture is carried out for 30 days, and the proliferation culture medium contains 0.2mg/L of indoleacetic acid, 0.25mg/L of 6-benzylaminoadenine and 0.5mg/L of kinetin;
4) Culturing primary seedling cluster buds by using a strong seedling culture medium to obtain strong seedlings of centella asiatica, wherein the culture temperature is 24-26 ℃, the illumination intensity is 2000lux, the illumination time is 12 hours/day, the culture is 10 days, the illumination is performed under the alternate environment of white light and red and blue light, the illumination is performed for 8 hours/day, the illumination is performed for 4 hours/day, the dark culture is performed for 12 hours/day, the circulation is sequentially performed, the red light and blue light ratio is 2:3, the strong seedling culture medium contains 0.3mg/L of 6-benzylaminoadenine, 0.3mg/L of indoleacetic acid, 0.5g/L of activated carbon, and 0.2g/L of hydrolyzed casein and 20 mu mol/L of methyl jasmonate;
5) Culturing strong seedlings of centella asiatica by using a rooting culture medium until roots grow to 2-4cm, obtaining the seedlings of centella asiatica with roots, wherein the culture temperature is 24-26 ℃, the illumination intensity is 2000lux, the illumination time is 10-12 hours/day, the culture is 21 days, and the rooting culture medium contains 0.3mg/L of indoleacetic acid, 0.25mg/L of naphthylacetic acid and 0.75g/L of activated carbon;
6) Transplanting the centella asiatica with root seedlings after hardening the seedlings into a matrix for culture, wherein the transplanted matrix consists of seedling raising soil, sheep manure and vermiculite according to the mass ratio of 5:1:1;
the induction medium, the proliferation medium and the strong seedling medium are all MS culture medium, the rooting medium is 1/2MS culture medium, and the induction medium, the proliferation medium, the strong seedling medium and the rooting medium all contain 5.0g/L agar and 30g/L sucrose, and the pH value is 5.8.
2. The rapid propagation method of centella asiatica tissue culture according to claim 1, wherein the sterilization process in step 1) is specifically: sequentially soaking the stems with axillary buds of centella asiatica with aqueous solution of liquid detergent with mass concentration of 1% for 5-10min, washing with tap water for 15-30min, washing with sterile water once, soaking with alcohol with volume concentration of 75% for 30-50s, sterilizing with 150ml of 0.1% mercuric chloride added with 2-3 drops of Tween-80 for 6-8min, washing with sterile water for 3-5 times, and spreading on sterilized filter paper to remove surface moisture.
3. The rapid propagation method of centella asiatica tissue culture according to claim 1, wherein the specific process of hardening off in the step 6) is as follows: removing centella asiatica with root seedling together with culture flask, transferring into greenhouse, standing at 25-27deg.C for 7 days, opening bottle cap, adding tap water, and hardening off seedling for 3-4 days.
4. The method for rapid propagation of centella asiatica tissue culture according to claim 3, wherein the plastic film is used for moisturizing 7-10 days after transplanting, a layer of newspaper is covered on the plastic film, the shading rate is 60% -70%, the air humidity is 85% -90%, and spraying is carried out 1 time a day.
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