CN115226630B - Tissue culture propagation method for artemisia rupestris L - Google Patents
Tissue culture propagation method for artemisia rupestris L Download PDFInfo
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- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
- A01H4/00—Plant reproduction by tissue culture techniques ; Tissue culture techniques therefor
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
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- A01H4/00—Plant reproduction by tissue culture techniques ; Tissue culture techniques therefor
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Abstract
The invention relates to the field of medicinal plant tissue culture, and particularly discloses a method for propagating Artemisia rupestris L.var.media by tissue culture, which comprises the following steps: culturing sterile stem segments with buds of the middle Asia artemisia selengensis in a rooting culture medium, and propagating test-tube plantlets of the middle Asia artemisia selengensis; the rooting culture medium is a solid culture medium obtained by adding naphthylacetic acid, cane sugar and a coagulant into a solid MS basic culture medium, wherein the content of the naphthylacetic acid in the rooting culture medium is 0.4-0.6mg/L, and the content of the cane sugar in the rooting culture medium is 30g/L. The tissue culture and propagation method of the artemisia scoparia can quickly propagate a large number of artemisia scoparia tissue culture seedlings in a short time by using a small amount of plant materials, and the method is used for production research. All the bred plantlets obtained by the method are from the same plant, the variety and the genetic character are consistent, and meanwhile, the method is convenient for providing a foundation for the genetic transformation related research of the Artemisia rupestris L.
Description
Technical Field
The invention relates to the field of medicinal plant tissue culture, in particular to a method for propagating artemisia rupestris L by tissue culture.
Background
Artemisia absinthium (Artemisia absinthium) is a perennial herb of Artemisia of Compositae, and is widely distributed in central or central Asia, european countries, northern Africa, canada in North Africa and North America, and eastern USA. It is distributed in Xinjiang in China, and has a small amount of cultivation in Nanjing and Shandong. It has an edible history of about 300 years as a main raw material of "wormwood wine" well known in europe and has a long medicinal history, and is often used for treating hepatitis, dyspepsia, stomachache, anemia, anorexia, and the like in europe, countries such as kashmir, iran, india, and pakistan. Modern pharmacological research shows that the medicine has the activities of antibiosis, antioxidation, liver protection, neuroprotection, depression resistance and the like. The extract is a cosmetic raw material, and has been developed into numerous cosmetics on the market. With the increasing demand of the middle-Asia artemisia selengensis in the fields of wine brewing, cosmetics and medicines and health care products, the large-scale breeding of the middle-Asia artemisia selengensis becomes an important industrial problem which needs to be solved urgently. Therefore, in order to solve the problems of scale breeding and introduction of the artemisia rupestris L, a rapid tissue culture and propagation method of the artemisia rupestris L is urgently needed.
Disclosure of Invention
The invention provides a high-quality, rapid and efficient method for tissue culture and rapid propagation of middle-Asia artemisia selengensis, aiming at the problems of introduction of the middle-Asia artemisia selengensis as a new resource and resource scarcity, and directly inducing the stem end of the bud of the middle-Asia artemisia selengensis to directly root and grow into a seedling in one step.
In order to solve the technical problem, the invention provides a method for tissue culture and propagation of artemisia rupestris, which comprises the following steps: culturing sterile stem segments with buds of the middle Asia artemisia selengensis in a rooting culture medium, and propagating tissue culture seedlings of the middle Asia artemisia selengensis; the rooting culture medium is a solid culture medium obtained by adding naphthylacetic acid, sucrose and a coagulant into a solid MS basic culture medium, wherein the content of the naphthylacetic acid in the rooting culture medium is 0.4-0.6mg/L, and the content of the sucrose in the rooting culture medium is 30g/L.
The pH value of the rooting culture medium is 6.2-6.5.
The coagulating agent of the rooting culture medium is agar, and the content of the agar in the rooting culture medium is 7-9g/L.
The method comprises the step of obtaining the sterile stem section with buds of the artemisia intermedia, wherein the step of disinfection comprises the step of immersing an explant into a mixed solution of 0.1% mercuric chloride and 0.04-0.06% tween for 6-7 min. The preferred method comprises the step of immersing the explant in a mixture of 0.1% mercuric chloride +0.05% tween for 6 min.
The stem segment length of the sterile stem segment with buds of the middle Asia Artemisia selengensis is 1.0-1.5cm.
The sterile stem segment with buds of the artemisia rupestris L is from A1 or A2:
a1, collecting stems of middle-Asia artemisia selengensis which grows vigorously and does not have diseases and insect pests, cutting the stems into stem sections with buds, and sterilizing to obtain sterile stem sections with the buds of the middle-Asia artemisia selengensis;
a2, cutting the stem of the tissue culture seedling of the middle-Asia artemisia selengensis obtained through tissue culture into stem sections with buds to obtain sterile stem sections with the buds of the middle-Asia artemisia selengensis.
And (4) culturing for 25-30 days.
The culture conditions of the culture are that the temperature is 23-26 ℃, the relative humidity is 50-70%, the illumination is 12-14 h/10-12 h dark every day, and the illumination intensity is 1000-3000lux.
The tissue culture and propagation expanding method of artemisia anomala also comprises the step of transplanting and planting after hardening off the tissue culture seedlings of artemisia anomala.
The tissue culture and propagation method of the artemisia rupestris L can rapidly propagate a large number of seedlings of the artemisia rupestris L in a short time by using a small amount of plant materials, and is used for production research. According to the tissue culture seedling obtained by the method, all the bred seedlings come from the same plant, the variety and the genetic character are consistent, and meanwhile, a foundation is provided for the genetic transformation related research of the artemisia scoparia.
Detailed Description
The present invention is described in further detail below with reference to specific embodiments, which are given for the purpose of illustration only and are not intended to limit the scope of the invention. The examples provided below serve as a guide for further modifications by a person skilled in the art and do not constitute a limitation of the invention in any way.
The experimental procedures in the following examples, unless otherwise specified, were carried out in a conventional manner according to the techniques or conditions described in the literature in this field or according to the product instructions. Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.
Artemisia scoparia in the following examples is commercially available.
Preliminary experiments
Screening experiment of disinfection mode of tissue culture explant of Artemisia sieboldii Miq:
collecting branches of Artemisia capillaris Thunb in the noon or afternoon of sunny weather, cleaning, selecting stem segments with buds, cutting into segments with length of 1.0-1.5cm, and obtaining stem segments with buds as explants for use.
3 explant disinfection modes are set, and the disinfection modes are as follows:
the disinfection mode 1: and (3) placing the explant obtained in the step (1) under running water for washing for 30min for preliminary sterilization treatment for later use. After 30min of superclean ultraviolet sterilization, the remaining operations were treated in a superclean bench. Immersing the washed explant in an ethanol solution with the volume percentage content of 75% (v/v) for 30s, then immediately taking out, washing for 3 times by using sterile water (an autoclave is sterilized at 121 ℃ for 15 min), immersing the explant in a mixed solution of 0.1% mercuric chloride and 0.05% tween for 6min, and finally washing for 5 times by using the sterile water to obtain a sterile stem section with buds for later use.
And (3) a disinfection mode 2: and (3) placing the explant obtained in the step (1) under running water for washing for 30min for preliminary sterilization treatment for later use. After 30min of ultra-clean bench ultraviolet sterilization, immersing the washed explant in an ethanol solution with the volume percentage content of 75% (v/v) for 30s, then immediately taking out, washing for 3 times by using sterile water (sterilized by an autoclave at 121 ℃ for 15 min), immersing the explant in a mixed solution of 0.1% mercuric chloride and 0.05% tween for 8min, and finally washing for 5 times by using sterile water to obtain a stem section with a germ-free area for later use.
And (3) a disinfection mode: and (4) placing the explants obtained in the step (1) in running water to wash for 30min for preliminary sterilization treatment for later use. After 30min of ultra-clean bench ultraviolet sterilization, immersing the washed explant in 75% (v/v) ethanol solution by volume percent for 30s, then immediately taking out, washing with sterile water (autoclave is sterilized at 121 ℃ for 15 min) for 3 times, immersing the explant in a mixed solution of 0.1% mercuric chloride and 0.15% tween for 6min, and finally washing with sterile water for 5 times to obtain a sterile stem section with buds for later use.
The obtained sterilized explants were inoculated into primary medium. The primary culture medium is a culture medium which takes a solid MS basic culture medium as a basic culture medium, the concentration of 6-BA (6-benzylaminopurine) is 2.0mg/L, the concentration of sucrose is 30g/L, the concentration of agar is 9g/L, and the pH value is 6.2. The specific preparation method of the primary culture medium (taking 1L as an example) can be as follows: dissolving 4.48 g of MS culture medium basic salt (Phytotechnology company, HGY 0519284A), 30g of cane sugar (national drug group chemical reagent company, 20211118) and 9g of agar powder (Beijing Ding Guosheng biotechnology company, B214101300) in 2000mL conical bottles (cleaned and rinsed with deionized water for 3 times), adding 6-BA with the final concentration of 2.0mg/L, adding water to a constant volume of 1L, adjusting the pH value to 6.2, carrying out autoclaving at 121 ℃ for 15min, subpackaging the bottles in a super clean bench while hot after sterilization is finished, and inoculating the tissue culture seedlings treated in different sterilization modes into the bottles after the tissue culture seedlings are cooled. Sealing the tissue culture bottle, and culturing in a tissue culture room.
In the above steps, 3 seedlings were inoculated per bottle, 10 bottles were inoculated per treatment, and 3 times were repeated. The culture conditions include 23-26 deg.C, relative humidity of 50-70%, illumination intensity of 1000-3000lux, illumination for 12 hr, and darkness for 12 hr.
The contamination rates of the treatments using the sterilization mode 1, the sterilization mode 2 and the sterilization mode 3 were counted, and the results are shown in table 1:
TABLE 1 explant contamination rates for different disinfection regimes
| Mode of disinfection | Infection rate/% |
| Disinfection mode 1 | 27％ |
| Disinfection mode 2 | 80％ |
| Disinfection mode 3 | 83％ |
By comparing the disinfection mode 1 with the disinfection mode 2, it can be concluded that when the explant is immersed in the mixed solution of 0.1% mercuric chloride and 0.05% tween for disinfection, the explant is immersed for 6min, the disinfection effect is better, the time is increased, and the pollution rate is increased. By comparing the disinfection mode 1 and the disinfection mode 3, it can be concluded that the disinfection effect is better when the concentration of tween in the mixed solution of mercuric chloride and tween is 0.05%, and the pollution rate is increased when the concentration is increased to 0.15%. In conclusion, under the condition that other conditions are not changed, the explant is immersed into the mixed solution of 0.1% mercuric chloride and 0.05% tween for 6min, so that the disinfection effect is better.
Example 1
The tissue culture propagation method of the artemisia rupestris L comprises the following steps:
step 1: selection of explant material
Collecting branches of Artemisia rupestris in the noon or afternoon of sunny weather, cleaning, selecting stem segments with buds, cutting into segments with length of 1.0-1.5cm, and obtaining stem segments with buds as explants for later use.
And 2, step: disinfection of explants
Disinfection mode (i.e. disinfection mode 1 in preliminary experiment): and (3) placing the explant obtained in the step (1) under running water for washing for 30min for preliminary sterilization treatment for later use. After 30min of superclean ultraviolet sterilization, the remaining operations were treated in a superclean bench. Immersing the washed explant in an ethanol solution with the volume percentage content of 75% (v/v) for 30s, then immediately taking out, washing for 3 times by using sterile water (the autoclave is sterilized at 121 ℃ for 15 minutes), immersing the explant in a mixed solution of 0.1% mercuric chloride and 0.05% tween for 6min, and finally washing for 5 times by using the sterile water to obtain a sterile stem section with buds for later use.
And step 3: rooting culture
Setting 5 rooting cultures, which are as follows:
rooting medium 1: the rooting medium 1 is a medium which takes a solid MS basic medium as a basic medium, the concentration of NAA (naphthylacetic acid) is 0.5mg/L, the concentration of sucrose is 30g/L, the concentration of agar is 9g/L, and the pH value is 6.2. The specific preparation method of the rooting medium 1 (taking 1L as an example) can be as follows: dissolving 2.24g of MS medium basic salt (Phyto technology company, HGY 0519284A), 30g of sucrose (national drug group chemical reagent company, 20211118) and 9g of agar powder (B214101300) in a 2000mL conical flask (cleaned and rinsed 3 times with deionized water), adding NAA with a final concentration of 0.5mg/L, adding water to a constant volume of 1L, adjusting the pH value to 6.2, autoclaving at 121 ℃ for 15min, subpackaging the sterilized solution in a tissue culture bottle while hot in a superclean bench, and cooling.
Rooting medium 2: the rooting culture medium 2 is a culture medium which takes a solid MS basic culture medium as a basic culture medium, the concentration of NAA (naphthylacetic acid) is 1.0mg/L, the concentration of sucrose is 30g/L, the concentration of agar is 9g/L, and the pH value is 6.2. The specific preparation method of the rooting medium 2 (taking 1L as an example) can be as follows: dissolving 2.24g of MS culture medium basic salt, 30g of cane sugar and 9g of agar powder in a 2000mL conical flask (cleaned and rinsed for 3 times by deionized water), adding 1.0mg/L NAA (N-acetyl-D-alanine) into the solution, adding water to a constant volume of 1L, adjusting the pH value to 6.2, carrying out autoclaving at 121 ℃ for 15min, filling the solution into a tissue culture bottle in a clean bench while the solution is hot after the sterilization is finished, and cooling the tissue culture bottle.
Rooting medium 3: the rooting medium 3 is a medium which takes a solid MS minimal medium as a basal medium, the concentration of NAA (naphthylacetic acid) is 1.5mg/L, the concentration of sucrose is 30g/L, the concentration of agar is 9g/L, and the pH value is 6.2. The specific preparation method of the rooting medium 3 (taking 1L as an example) can be as follows: dissolving 2.24g of MS culture medium basic salt, 30g of cane sugar and 9g of agar powder in a 2000mL conical flask (cleaned and rinsed for 3 times by deionized water), adding 1.5mg/L NAA (N-acetyl-D) of final concentration, adding water to a constant volume of 1L, adjusting the pH value to 6.2, carrying out autoclaving at 121 ℃ for 15min, filling the mixture into a tissue culture bottle in a superclean bench while the mixture is hot after the sterilization is finished, and cooling the tissue culture bottle.
Rooting medium 4: the rooting medium 4 is a medium which takes a solid MS basic medium as a basic medium, the concentration of IAA (indole-3-acetic acid) is 0.5mg/L, the concentration of sucrose is 30g/L, the concentration of agar is 9g/L, and the pH value is 6.2. The specific preparation method of the rooting medium 4 (taking 1L as an example) can be as follows: dissolving 2.24g of MS culture medium basic salt, 30g of cane sugar and 9g of agar powder in a 2000mL conical flask (cleaned and rinsed for 3 times by deionized water), adding IAA with the final concentration of 0.5mg/L, adding water to a constant volume of 1L, adjusting the pH value to 6.2, carrying out autoclaving at 121 ℃ for 15min, filling the mixture into a tissue culture bottle in a superclean bench while the mixture is hot after the sterilization is finished, and cooling the tissue culture bottle.
Rooting medium 5: the rooting culture medium 5 is a culture medium which takes a solid MS basic culture medium as a basic culture medium, the concentration of NAA (naphthylacetic acid) is 0.5mg/L, the concentration of IAA (indole-3-acetic acid) is 0.5mg/L, the concentration of sucrose is 30g/L, the concentration of agar is 9g/L, and the pH value is 6.2. The specific preparation method of the rooting medium 5 (taking 1L as an example) can be as follows: dissolving 2.24g of MS culture medium basic salt, 30g of cane sugar and 9g of agar powder in a 2000mL conical flask (cleaned and rinsed for 3 times by deionized water), adding NAA with the final concentration of 0.5mg/L and IAA with the final concentration of 0.5mg/L, adding water to the mixture to reach a constant volume of 1L, adjusting the pH value to 6.2, carrying out autoclaving at 121 ℃ for 15min, and then, immediately packaging the mixture in a tissue culture bottle in a clean bench and cooling the tissue culture bottle.
And (3) respectively inoculating the sterile stem segments with buds obtained in the step (2) into a rooting culture medium 1, a rooting culture medium 2, a rooting culture medium 3, a rooting culture medium 4 and a rooting culture medium 5, sealing the tissue culture bottle, and placing the sealed tissue culture bottle in a tissue culture room for culture.
In the above steps, 3 seedlings were inoculated per bottle, 3 bottles were inoculated per treatment, and 3 times of repetition. The culture conditions include 23-26 deg.C, relative humidity of 50-70%, illumination intensity of 1000-3000lux, illumination for 12 hr, and darkness for 12 hr.
The rooting time of the treatments using rooting medium 1, rooting medium 2, rooting medium 3, rooting medium 4 and rooting medium 5, and the rooting rate and root length at 30 days of culture were counted, and the results are shown in table 2:
TABLE 2 rooting indicators for different rooting media
By comparing the rooting medium 1 with the rooting medium 2 and the rooting medium 3, it can be concluded that the addition of 0.5mg NAA in the rooting medium helps to promote rooting, shorten the rooting time, and promote root growth. By comparing rooting medium 1 and rooting medium 4, it can be concluded that addition of IAA to the medium also induced rooting, but the overall effect was significantly less than NAA. By comparing rooting medium 1 and rooting medium 5, it can be concluded that the addition of NAA in rooting medium promotes rooting, but the overall effect is not as good as 0.5mg/L NAA alone. In conclusion, the method is simple, the cost is saved, the rooting rate is high, and the rooting time is shortened by independently adding 0.5mg/L of NAA growth hormone.
And 4, step 4: hardening and transplanting seedlings:
firstly, preparing a planting matrix (plant carbon ash: perlite =3, volume ratio is 1), placing the planting matrix in an autoclave at 121 ℃ for sterilization for 60min, and cooling the mixed matrix for later use; observing the tissue culture seedlings, opening a tissue culture bottle when the tissue culture seedlings grow to 8-10cm higher than the overground part, exposing the tissue culture bottle in a 50% shading environment for hardening the seedlings, cleaning a culture medium at the root after hardening the seedlings for 3-5 days, and transferring the tissue culture seedlings to a mixed matrix for planting.
The present invention has been described in detail above. It will be apparent to those skilled in the art that the invention can be practiced in a wide range of equivalent parameters, concentrations, and conditions without departing from the spirit and scope of the invention and without undue experimentation. While the invention has been described with reference to specific examples, it will be appreciated that the invention may be further modified. In general, this application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure as come within known or customary practice within the art to which the invention pertains. The use of some of the essential features is possible within the scope of the claims attached below.
Claims (6)
1. A method for tissue culture and propagation of Artemisia rupestris L is characterized by comprising the following steps: culturing sterile stem segments with buds of the middle Asia artemisia selengensis in a rooting culture medium, and propagating tissue culture seedlings of the middle Asia artemisia selengensis; the rooting culture medium is MS minimal medium, 0.4-0.6mg/L naphthylacetic acid, 30g/L sucrose and 7-9g/L agar, and the pH value is 6.2-6.5;
the step of obtaining the sterile budded stem segment of the artemisia scoparia comprises disinfection, wherein the disinfection comprises the step of immersing the explant into a mixed solution of 0.1% of mercury bichloride and 0.05% of tween for 6 min.
2. The method according to claim 1, wherein the stem segment of the sterile sprouted stem segment of Artemisia intermedia is 1.0-1.5cm in length.
3. The method according to any one of claims 1 to 2, wherein the sterile sprouted stem segment of artemisia intermedia is from A1 or A2:
a1, collecting stems of middle-Asia artemisia selengensis which grows vigorously and does not have diseases and insect pests, cutting the stems into stem sections with buds, and sterilizing to obtain sterile stem sections with the buds of the middle-Asia artemisia selengensis;
a2, cutting stems of tissue culture seedlings of the middle Asia artemisia selengensis obtained through tissue culture into stem sections with buds to obtain sterile stem sections with the buds of the middle Asia artemisia selengensis.
4. The method of claim 1, wherein the culturing is in rooting medium for 25-30 days.
5. The method according to claim 1, wherein the culturing is carried out under conditions of a temperature of 23-26 ℃, a relative humidity of 50-70%, a light intensity of 12-14 hours per day/a dark intensity of 10-12 hours per day, and a light intensity of 1000-3000lux.
6. The method of claim 1, further comprising acclimatizing the tissue culture plantlets of artemisia scoparia and planting the acclimatized plantlets in a transplanting manner.
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