CN111066657B - Tissue culture method for sophora flavescens - Google Patents

Tissue culture method for sophora flavescens Download PDF

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CN111066657B
CN111066657B CN202010053737.5A CN202010053737A CN111066657B CN 111066657 B CN111066657 B CN 111066657B CN 202010053737 A CN202010053737 A CN 202010053737A CN 111066657 B CN111066657 B CN 111066657B
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CN111066657A (en
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罗宇东
胡琦敏
谭安蔷
庞晓云
邹意华
李娜
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Guangxi University Of Chinese Medicine Pharmaceutical Factory
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    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
    • A01H4/00—Plant reproduction by tissue culture techniques ; Tissue culture techniques therefor
    • A01H4/001—Culture apparatus for tissue culture
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
    • A01H4/00—Plant reproduction by tissue culture techniques ; Tissue culture techniques therefor
    • A01H4/008—Methods for regeneration to complete plants
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
    • A01H6/00—Angiosperms, i.e. flowering plants, characterised by their botanic taxonomy
    • A01H6/20—Brassicaceae, e.g. canola, broccoli or rucola

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Abstract

The invention discloses a tissue culture method of sophora flavescens ait, which comprises the following steps: sterilizing explants, germinating and culturing seeds, differentiating and culturing cluster buds, culturing rooting and strengthening seedlings and planting. The plant growth regulators with different concentrations are used in different development stages of the radix sophorae flavescentis, so that the quantity and the quality of buds, differentiated buds and seedlings of the radix sophorae flavescentis are obviously improved, and the survival rate of tissue culture is improved.

Description

Tissue culture method for vine radix sophorae flavescentis
Technical Field
The invention relates to the technical field of tissue culture and propagation, in particular to a tissue culture method of radix sophorae flavescentis.
Background
Radix Sophorae Flavescentis, radix Stemonae and radix Sophorae Flavescentis, which is one of the main drugs in YA YAJIAO HAIDU powder, is the dry root of herba Eichhorniae saddle of the genus Ecliptae of the family Asclepiadaceae, and is widely used in Yunnan and Guangxi provinces for treating dysentery, diarrhea due to damp-heat, heart and stomach pain, common cold and fever, chronic gastritis, traumatic injury, etc.
In the prior art, the propagation method of the vine radix sophorae flavescentis generally adopts seed propagation, layering propagation, tissue culture and the like; the propagation of the sophora vine seeds is time-consuming and labor-consuming, seedlings can emerge in 30 to 40 days, the seedlings can be transplanted after 2 to 3 years, and the time for growing the sophora vine seeds into medicinal materials is longer. The survival rate of layering propagation of the radix sophorae flavescentis is low, and standardized planting is not facilitated. The tissue culture (rapid propagation) efficiency of the vine radix sophorae flavescentis is high, and the tissue culture method is simple, convenient, easy, quick in effect taking and good in effect, so that the tissue culture method is recommended to be adopted in production. Once the tissue culture seedling of the sophora flavescens is survived, the drought resistance is strong after the root is deeply pricked, and the environment survival ability is strong.
Therefore, how to provide a tissue culture method capable of significantly improving the quality of a tissue culture seedling of radix sophorae flavescentis is a technical problem to be solved urgently by the technical personnel in the field.
Disclosure of Invention
In view of this, the invention provides a method for tissue culture of sophora moorcroftiana, which uses plant growth regulators with different concentrations at different development stages of sophora moorcroftiana, so as to significantly improve the number and quality of buds, differentiated buds and seedlings of sophora moorcroftiana, and improve the survival rate of tissue culture.
In order to achieve the purpose, the invention adopts the following technical scheme:
a tissue culture method of radix sophorae flavescentis comprises the following steps:
1) And (3) explant sterilization: pretreating the fruit pods of the radix sophorae flavescentis, taking out seeds in the fruit pods, and disinfecting the seeds for later use;
the process of pretreating the fruit pods of radix sophorae flavescentis and disinfecting the seeds comprises the following steps:
11 Washing the fruit pod of radix Sophorae Flavescentis with sterile water for 3 times, each for 1-2 min;
12 Taking out the seeds in the fruit pods of the vine and lightyellow sophora root, soaking the seeds in 75% alcohol for 20-30s, and then washing the seeds with sterile water for 3 times, each time for 1-2 minutes;
13 Soaking the seeds treated in the step 12) with 0.1 percent mercuric chloride for 10-15min, and then washing with sterile water for 4-5 times, 1min each time for later use;
2) Seed germination culture: inoculating the seeds sterilized in the step 1) into an MS culture medium added with 0.8-2.0mg/L6-BA, 0.2-0.5mg/L LNAA and 1g/L active carbon for germination culture, and obtaining vine sophora flavescens sprouts after 20-25 days;
3) And (3) differentiated culture of cluster buds: inoculating the stem lightyellow sophora root sprouts obtained in the step 2) into an MS culture medium added with 3.0-4.0mg/L6-BA and 0.2-0.5mg/LNAA for differential culture, and obtaining stem lightyellow sophora root differentiated sprouts after 35-45 days;
4) Rooting and seedling strengthening culture: when the stem sophora flavescens differentiated bud in the step 3) grows into 3-4cm seedlings, transferring the stem sophora flavescens differentiated bud into an MS culture medium added with 1.0mg/LNAA and 1g/L of activated carbon to carry out rooting and seedling strengthening culture, and obtaining rooted seedlings after 45-55 days;
5) Hardening and planting: culturing the rooted seedlings obtained in the step 4) in natural light for 3 days, opening the bottle caps of the test tube seedlings, hardening the seedlings for 3 days, then slightly clamping the test tube seedlings by using forceps, washing off agar at the base part, transplanting the test tube seedlings into sandy soil added with 1/2MS nutrient solution, transplanting the test tube seedlings into soil after 4 weeks, and performing field planting.
The technical effect achieved by the technical scheme is as follows: in the tissue culture, the reasonable matching of hormone and auxin is the key for achieving the optimal culture effect, and in the stages of seed germination, cluster bud differentiation, rooting and seedling strengthening, plant growth regulators with different concentrations are added into an MS solid culture medium, so that 6-BA and NAA with specific concentrations have a synergistic interaction effect, the germination rate and cluster bud differentiation amount are obviously improved, and the seedling quality is improved;
in the process of plant rooting culture, the light irradiation time is properly increased to be beneficial to the rooting of the plantlets, but the direct irradiation of the roots by strong light can inhibit the growth of the roots, so that the active carbon is added into the rooting and strong seedling culture medium to provide a dark rooting environment and prevent browning; the active carbon can also adsorb some harmful substances released in the growth process of plants, and is beneficial to the growth of seedlings.
The activated carbon can adsorb some harmful substances released in the growth process of plants, so that the seedlings grow strongly by adding the activated carbon in the rooting and strong seedling culture stage in the experiment.
As a preferable technical scheme of the invention, in the steps 2) -4), the temperature of the culture room is 23-27 ℃, the illumination intensity is 1000-1200lx, and the illumination is 12-14h every day.
The technical effect achieved by the technical scheme is as follows: and (3) promoting the rooting and seedling growing of the plant at proper temperature, illumination intensity and time, and otherwise, inhibiting the production of the culture tissue by strong illumination if the temperature is too high or too low.
As a preferred technical scheme of the invention, in the steps 2) to 4), the pH value of the MS culture medium is controlled to be 6.2 to 6.7 (measured by a precision pH test paper).
According to the technical scheme, compared with the prior art, the tissue culture method for the radix sophorae flavescentis provided by the invention has the advantages that the germination rate, the growth amount of the buds, the differentiation amount of the cluster buds, the number and the quality of seedlings of the radix sophorae flavescentis seeds are obviously improved;
moreover, stem tips, cotyledons, leaves and the like are selected as the explants for tissue culture of other plants in the same family, the stems and leaves of the bittersweet root have milk and are densely covered with villi, and the explant is selected in consideration that the stem tips are taken and are not easy to sterilize, and the injury of the explant can release the milk to influence the survival rate of the explant after being inoculated to a culture medium and the like.
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In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the embodiments or the prior art descriptions will be briefly described below, it is obvious that the drawings in the following description are only embodiments of the present invention, and for those skilled in the art, other drawings can be obtained according to the provided drawings without creative efforts.
FIG. 1 is a schematic diagram showing the growth of shoot of Sophora flavescens ait in step 2) of example 12;
FIG. 2 is a schematic view showing the growth of differentiated shoot of Sophora flavescens Aiton in step 3) of example 12;
FIG. 3 is a schematic view of the growth of the seedling of Sophora flavescens Aiton in step 4) of example 12.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Example 1
A tissue culture method of radix sophorae flavescentis comprises the following steps:
1) And (3) explant sterilization: washing the vine and lightyellow sophora root fruit pods with sterile water; taking out the seeds in the fruit pods of the vine and lightyellow sophora root, soaking the seeds in 75% alcohol for 20s, and then washing the seeds with sterile water for 1 time; soaking the treated seeds with 0.1% mercuric chloride for 10min, and washing with sterile water for 4 times;
2) Seed germination culture: inoculating the seeds sterilized in the step 1) into an MS culture medium added with 0.8mg/L6-BA, 0.5mg/L LNAA and 1g/L active carbon for germination culture, controlling the pH value of the MS culture medium to be 6.2, controlling the temperature of a culture room to be 23 ℃, controlling the illumination intensity to be 1000lx, and illuminating for 12h and 25d every day to obtain the premna microphylla;
3) And (3) differentiated culture of cluster buds: inoculating the premna lobata sprouts obtained in the step 2) into an MS culture medium added with 3.0mg/L6-BA and 0.2mg/LNAA to perform differential culture, controlling the pH value of the MS culture medium to be 6.2, controlling the temperature of a culture room to be 23 ℃, controlling the illumination intensity to be 1000lx, and illuminating for 12h and 45d every day to obtain premna lobata sprouts;
4) Rooting and seedling strengthening culture: when the stem sophora flavescens differentiated bud in the step 3) grows into 3cm seedlings, transferring the stem sophora flavescens differentiated bud into an MS culture medium added with 1.0mg/LNAA and 1g/L activated carbon to carry out rooting and seedling strengthening culture, controlling the pH value of the MS culture medium to be 6.2, controlling the temperature of a culture room to be 23 ℃, the illumination intensity to be 1000lx, illuminating for 12h every day, and obtaining rooting seedlings after 55d;
5) Hardening and planting: culturing the rooted seedlings obtained in the step 4) in natural light for 3 days, opening the bottle caps of the test tube seedlings, hardening the seedlings for 3 days, then slightly clamping the test tube seedlings by using forceps, washing off agar at the base part, transplanting the test tube seedlings into sandy soil added with 1/2MS nutrient solution, transplanting the test tube seedlings into soil after 4 weeks, and performing field planting.
Example 2
A tissue culture method of radix sophorae flavescentis comprises the following steps:
1) And (3) explant sterilization: washing the vine and lightyellow sophora root fruit pods with sterile water; taking out the seeds in the fruit pods of the vine and lightyellow sophora root, soaking the seeds in 75% alcohol for 30s, and then washing the seeds with sterile water for 1 time; soaking the treated seeds with 0.1% mercuric chloride for 15min, and washing with sterile water for 5 times;
2) Seed germination culture: inoculating the seeds sterilized in the step 1) into an MS culture medium added with 2.0mg/L6-BA, 0.5mg/L LNAA and 1g/L active carbon for germination culture, controlling the pH value of the MS culture medium to be 6.7, controlling the temperature of a culture room to be 27 ℃, and the illumination intensity to be 1200lx, and obtaining the premna microphylla sprouts after illumination for 14h and 22d every day;
3) And (3) differentiated culture of cluster buds: inoculating the stem sophora flavescens sprouts obtained in the step 2) into an MS culture medium added with 3.5mg/L6-BA and 0.2mg/LNAA to perform differential culture, controlling the pH value of the MS culture medium to be 6.7, controlling the temperature of a culture room to be 27 ℃, controlling the illumination intensity to be 1200lx, illuminating for 14h every day, and obtaining the stem sophora flavescens differentiated sprouts after 40d;
4) Rooting and seedling strengthening culture: when the stem sophora flavescens differentiated bud in the step 3) grows into 4cm seedlings, transferring the stem sophora flavescens differentiated bud into an MS culture medium added with 1.0mg/LNAA and 1g/L activated carbon to carry out rooting and seedling strengthening culture, controlling the pH value of the MS culture medium to be 6.7, controlling the temperature of a culture room to be 27 ℃, the illumination intensity to be 1200lx, illuminating for 14h every day, and carrying out 50d to obtain rooting seedlings;
5) Hardening and planting: culturing the rooted seedlings obtained in the step 4) in natural light for 3 days, opening the caps of the test tube seedlings, hardening off the seedlings for 3 days, lightly clamping the test tube seedlings by using forceps, washing off agar at the base, transplanting the test tube seedlings into sandy soil added with 1/2MS nutrient solution, transplanting the test tube seedlings into soil after 4 weeks, and performing field planting.
Example 3
A tissue culture method of radix sophorae flavescentis comprises the following steps:
1) And (3) explant sterilization: washing the vine and lightyellow sophora root fruit pods with sterile water; taking out the seeds in the fruit pods of the sophora flavescens, soaking the seeds in 75% alcohol for 21s, and then washing the seeds with sterile water for 1 time; soaking the treated seeds with 0.1% mercuric chloride for 11min, and washing with sterile water for 5 times;
2) Seed germination culture: inoculating the seeds disinfected in the step 1) into an MS culture medium added with 0.8mg/L6-BA, 0.2mg/L LNAA and 1g/L active carbon for germination culture, controlling the pH value of the MS culture medium to be 6.3, controlling the temperature of a culture room to be 24 ℃, the illumination intensity to be 1050lx, and illuminating for 1 h and 23d every day to obtain sophora flavescens sprouts;
3) And (3) differentiated culture of cluster buds: inoculating the premna lobata sprouts obtained in the step 2) into an MS culture medium added with 4.0mg/L6-BA and 0.2mg/LNAA to perform differentiation culture, controlling the pH value of the MS culture medium to be 6.3, controlling the temperature of a culture room to be 24 ℃, controlling the illumination intensity to be 1050lx, illuminating for 13h every day, and obtaining premna lobata sprouts after 42d illumination;
4) Rooting and seedling strengthening culture: when the stem sophora flavescens differentiated buds in the step 3) grow into 4cm seedlings, transferring the stem sophora flavescens differentiated buds into an MS culture medium added with 1.0mg/LNAA and 1g/L activated carbon to perform rooting and strong seedling culture, controlling the pH value of the MS culture medium to be 6.3, controlling the temperature of a culture room to be 24 ℃, the illumination intensity to be 1050lx, illuminating for 13h every day, and after 51d, obtaining rooting seedlings;
5) Hardening and planting: culturing the rooted seedlings obtained in the step 4) in natural light for 3 days, opening the caps of the test tube seedlings, hardening off the seedlings for 3 days, lightly clamping the test tube seedlings by using forceps, washing off agar at the base, transplanting the test tube seedlings into sandy soil added with 1/2MS nutrient solution, transplanting the test tube seedlings into soil after 4 weeks, and performing field planting.
Example 4
A tissue culture method of radix Sophorae Flavescentis comprises the following steps:
1) And (3) explant sterilization: washing the vine and lightyellow sophora root fruit pods with sterile water; taking out the seeds in the fruit pods of the vine and lightyellow sophora root, soaking the seeds in 75% alcohol for 22s, and then washing the seeds with sterile water for 1 time; soaking the treated seeds with 0.1% mercuric chloride for 12min, and washing with sterile water for 4 times;
2) Seed germination culture: inoculating the seeds sterilized in the step 1) into an MS culture medium added with 1.0mg/L6-BA, 0.2mg/L LNAA and 1g/L active carbon for germination culture, controlling the pH value of the MS culture medium to be 6.4, controlling the temperature of a culture room to be 26 ℃, the illumination intensity to be 1100lx, illuminating for 14h every day, and obtaining the premna microphylla sprouts after 23d;
3) And (3) differentiating and culturing cluster buds: inoculating the premna lobata sprouts obtained in the step 2) into an MS culture medium added with 3.0mg/L6-BA and 0.3mg/LNAA to perform differentiation culture, controlling the pH value of the MS culture medium to be 6.4, controlling the temperature of a culture room to be 26 ℃, controlling the illumination intensity to be 1100lx, illuminating for 12h every day, and obtaining premna lobata sprouts after 38d;
4) Rooting and seedling strengthening culture: when the stem sophora flavescens differentiated bud in the step 3) grows into 4cm seedlings, transferring the stem sophora flavescens differentiated bud into an MS culture medium added with 1.0mg/LNAA and 1g/L activated carbon to carry out rooting and seedling strengthening culture, controlling the pH value of the MS culture medium to be 6.6, controlling the temperature of a culture room to be 25 ℃, the illumination intensity to be 1150lx, and illuminating for 13h and 53d every day to obtain rooting seedlings;
5) Hardening and planting: culturing the rooted seedlings obtained in the step 4) in natural light for 3 days, opening the caps of the test tube seedlings, hardening off the seedlings for 3 days, lightly clamping the test tube seedlings by using forceps, washing off agar at the base, transplanting the test tube seedlings into sandy soil added with 1/2MS nutrient solution, transplanting the test tube seedlings into soil after 4 weeks, and performing field planting.
Example 5
A tissue culture method of radix sophorae flavescentis comprises the following steps:
1) And (3) explant sterilization: washing the vine and lightyellow sophora root fruit pods with sterile water; taking out the seeds in the fruit pods of the sophora flavescens, soaking the seeds in 75% alcohol for 23s, and then washing the seeds with sterile water for 1 time; soaking the treated seeds with 0.1% mercuric chloride for 13min, and washing with sterile water for 5 times;
2) Seed germination culture: inoculating the seeds disinfected in the step 1) into an MS culture medium added with 1.5mg/L6-BA, 0.2mg/L LNAA and 1g/L active carbon for germination culture, controlling the pH value of the MS culture medium to be 6.5, controlling the temperature of a culture room to be 25 ℃, the illumination intensity to be 1200lx, and illuminating for 12h and 23d every day to obtain sophora flavescens sprouts;
3) And (3) differentiated culture of cluster buds: inoculating the premna lobata sprouts obtained in the step 2) into an MS culture medium added with 3.5mg/L6-BA and 0.3mg/LNAA to perform differentiation culture, controlling the pH value of the MS culture medium to be 6.2, controlling the temperature of a culture room to be 23 ℃, controlling the illumination intensity to be 1160lx, illuminating for 13h every day, and obtaining premna lobata sprouts after 42d illumination;
4) Rooting and seedling strengthening culture: when the stem sophora flavescens differentiated bud in the step 3) grows into 3cm seedlings, transferring the stem sophora flavescens differentiated bud into an MS culture medium added with 1.0mg/LNAA and 1g/L activated carbon to carry out rooting and seedling strengthening culture, wherein the pH value of the MS culture medium is controlled to be 6.2, the temperature of a culture room is 23 ℃, the illumination intensity is 1160lx, the illumination is 13h every day, and after 52d, the rooting seedlings are obtained;
5) Hardening and planting: culturing the rooted seedlings obtained in the step 4) in natural light for 3 days, opening the bottle caps of the test tube seedlings, hardening the seedlings for 3 days, then slightly clamping the test tube seedlings by using forceps, washing off agar at the base part, transplanting the test tube seedlings into sandy soil added with 1/2MS nutrient solution, transplanting the test tube seedlings into soil after 4 weeks, and performing field planting.
Example 6
A tissue culture method of radix sophorae flavescentis comprises the following steps:
1) And (3) explant sterilization: washing the vine and lightyellow sophora root fruit pods with sterile water; taking out the seeds in the fruit pods of the vine and lightyellow sophora root, soaking the seeds in 75% alcohol for 24s, and then washing the seeds with sterile water for 1 time; soaking the treated seeds with 0.1% mercuric chloride for 14min, and washing with sterile water for 4 times;
2) Seed germination culture: inoculating the seeds disinfected in the step 1) into an MS culture medium added with 2.0mg/L6-BA, 0.2mg/L LNAA and 1g/L active carbon for germination culture, controlling the pH value of the MS culture medium to be 6.5, controlling the temperature of a culture room to be 24 ℃, controlling the illumination intensity to be 1090lx, and illuminating for 12h and 22d every day to obtain sophora flavescens sprouts;
3) And (3) differentiated culture of cluster buds: inoculating the premna lobata sprouts obtained in the step 2) into an MS culture medium added with 4.0mg/L6-BA and 0.3mg/LNAA to perform differential culture, controlling the pH value of the MS culture medium to be 6.5, controlling the temperature of a culture room to be 24 ℃, controlling the illumination intensity to be 1090lx, and illuminating for 12h and 39d every day to obtain premna lobata sprouts;
4) Rooting and seedling strengthening culture: when the stem sophora flavescens differentiated bud in the step 3) grows into 4cm seedlings, transferring the stem sophora flavescens differentiated bud into an MS culture medium added with 1.0mg/LNAA and 1g/L activated carbon to carry out rooting and seedling strengthening culture, controlling the pH value of the MS culture medium to be 6.5, controlling the temperature of a culture room to be 24 ℃, the illumination intensity to be 1090lx, illuminating for 12h every day, and obtaining rooting seedlings after 48d;
5) Hardening and planting: culturing the rooted seedlings obtained in the step 4) in natural light for 3 days, opening the caps of the test tube seedlings, hardening off the seedlings for 3 days, lightly clamping the test tube seedlings by using forceps, washing off agar at the base, transplanting the test tube seedlings into sandy soil added with 1/2MS nutrient solution, transplanting the test tube seedlings into soil after 4 weeks, and performing field planting.
Example 7
A tissue culture method of radix Sophorae Flavescentis comprises the following steps:
1) And (3) explant sterilization: washing the vine and lightyellow sophora root fruit pods with sterile water; taking out the seeds in the fruit pods of the vine and lightyellow sophora root, soaking the seeds in 75% alcohol for 25s, and then washing the seeds with sterile water for 1 time; soaking the treated seeds with 0.1% mercuric chloride for 15min, and washing with sterile water for 5 times;
2) Seed germination culture: inoculating the seeds disinfected in the step 1) into an MS culture medium added with 0.8mg/L6-BA, 0.3mg/L LNAA and 1g/L active carbon for germination culture, controlling the pH value of the MS culture medium to be 6.7, controlling the temperature of a culture room to be 24 ℃, the illumination intensity to be 1170lx, and obtaining sophora flavescens sprouts after illumination for 13h and 21d every day;
3) And (3) differentiated culture of cluster buds: inoculating the stem sophora flavescens sprouts obtained in the step 2) into an MS culture medium added with 3.0mg/L6-BA and 0.5mg/LNAA to perform differentiation culture, controlling the pH value of the MS culture medium to be 6.7, controlling the temperature of a culture room to be 24 ℃, controlling the illumination intensity to be 1170lx, illuminating for 13h every day, and obtaining the stem sophora flavescens differentiated sprouts after 40d;
4) Rooting and seedling strengthening culture: when the stem sophora flavescens differentiated bud in the step 3) grows into 3-4cm seedlings, transferring the stem sophora flavescens differentiated bud into an MS culture medium added with 1.0mg/LNAA and 1g/L of activated carbon to carry out rooting and seedling strengthening culture, controlling the pH value of the MS culture medium to be 6.7, controlling the temperature of a culture room to be 24 ℃, controlling the illumination intensity to be 1170lx, and illuminating for 13h and 50d every day to obtain rooting seedlings;
5) Transplanting the rooted seedlings obtained in the step 4) into soil for field planting.
Example 8
A tissue culture method of radix sophorae flavescentis comprises the following steps:
1) And (3) explant sterilization: washing the vine and lightyellow sophora root fruit pods with sterile water; taking out the seeds in the fruit pods of the vine and lightyellow sophora root, soaking the seeds in 75% alcohol for 26s, and then washing the seeds with sterile water for 1 time; soaking the treated seeds with 0.1% mercuric chloride for 13min, and washing with sterile water for 4 times;
2) Seed germination culture: inoculating the seeds sterilized in the step 1) into an MS culture medium added with 1.0mg/L6-BA, 0.3mg/LNAA and 1g/L active carbon for germination culture, controlling the pH value of the MS culture medium to be 6.3, controlling the temperature of a culture room to be 25 ℃, the illumination intensity to be 1160lx, illuminating for 12h every day, and obtaining the premna microphylla sprouts after 24d;
3) And (3) differentiated culture of cluster buds: inoculating the premna lobata sprouts obtained in the step 2) into an MS culture medium added with 3.5mg/L6-BA and 0.5mg/LNAA to perform differentiation culture, controlling the pH value of the MS culture medium to be 6.3, controlling the temperature of a culture room to be 25 ℃, controlling the illumination intensity to be 1160lx, illuminating for 12h every day, and obtaining premna lobata sprouts after 37d;
4) Rooting and seedling strengthening culture: when the stem sophora flavescens differentiated bud in the step 3) grows into 3-4cm seedlings, transferring the stem sophora flavescens differentiated bud into an MS culture medium added with 1.0mg/LNAA and 1g/L of activated carbon to carry out rooting and seedling strengthening culture, controlling the pH value of the MS culture medium to be 6.3, controlling the temperature of a culture room to be 25 ℃, the illumination intensity to be 1160lx, illuminating for 12h every day, and after 45-55d, obtaining rooted seedlings;
5) Hardening and planting: culturing the rooted seedlings obtained in the step 4) in natural light for 3 days, opening the caps of the test tube seedlings, hardening off the seedlings for 3 days, lightly clamping the test tube seedlings by using forceps, washing off agar at the base, transplanting the test tube seedlings into sandy soil added with 1/2MS nutrient solution, transplanting the test tube seedlings into soil after 4 weeks, and performing field planting.
Example 9
A tissue culture method of radix sophorae flavescentis comprises the following steps:
1) And (3) explant sterilization: washing the vine and lightyellow sophora root fruit pods with sterile water; taking out the seeds in the fruit pods of the sophora flavescens, soaking the seeds in 75% alcohol for 27s, and then washing the seeds with sterile water for 1 time; soaking the treated seeds with 0.1% mercuric chloride for 13min, and washing with sterile water for 4 times;
2) Seed germination culture: inoculating the seeds disinfected in the step 1) into an MS culture medium added with 1.5mg/L6-BA, 0.3mg/L LNAA and 1g/L active carbon for germination culture, controlling the pH value of the MS culture medium to be 6.5, controlling the temperature of a culture room to be 25 ℃, the illumination intensity to be 1050lx, illuminating for 1 h every day, and obtaining sophora flavescens sprouts after 22d;
3) And (3) differentiated culture of cluster buds: inoculating the stem sophora flavescens sprouts obtained in the step 2) into an MS culture medium added with 4.0mg/L6-BA and 0.5mg/LNAA to perform differentiation culture, wherein the pH value of the MS culture medium is controlled to be 6.5, the temperature of a culture room is 25 ℃, the illumination intensity is 1050lx, and the stem sophora flavescens sprouts are obtained after illumination for 1 h and 37d every day;
4) Rooting and seedling strengthening culture: when the stem sophora flavescens differentiated bud in the step 3) grows into 3-4cm seedlings, transferring the stem sophora flavescens differentiated bud into an MS culture medium added with 1.0mg/LNAA and 1g/L of activated carbon to carry out rooting and seedling strengthening culture, controlling the pH value of the MS culture medium to be 6.5, controlling the temperature of a culture room to be 25 ℃, the illumination intensity to be 1050lx, and illuminating for 13h and 48d every day to obtain rooted seedlings;
5) Hardening and planting: culturing the rooted seedlings obtained in the step 4) in natural light for 3 days, opening the caps of the test tube seedlings, hardening off the seedlings for 3 days, lightly clamping the test tube seedlings by using forceps, washing off agar at the base, transplanting the test tube seedlings into sandy soil added with 1/2MS nutrient solution, transplanting the test tube seedlings into soil after 4 weeks, and performing field planting.
Example 10
A tissue culture method of radix Sophorae Flavescentis comprises the following steps:
1) And (3) explant sterilization: washing the vine and lightyellow sophora root fruit pods with sterile water; taking out the seeds in the fruit pods of the vine and lightyellow sophora root, soaking the seeds in 75% alcohol for 28s, and then washing the seeds with sterile water for 1 time; soaking the treated seeds with 0.1% mercuric chloride for 15min, and washing with sterile water for 5 times;
2) Seed germination culture: inoculating the seeds sterilized in the step 1) into an MS culture medium added with 2.0mg/L6-BA, 0.3mg/LNAA and 1g/L active carbon for germination culture, controlling the pH value of the MS culture medium to be 6.4, controlling the temperature of a culture room to be 26 ℃, the illumination intensity to be 1200lx, and obtaining sophora flavescens sprouts after illumination for 14h and 21d every day;
3) And (3) differentiating and culturing cluster buds: inoculating the stem sophora flavescens sprouts obtained in the step 2) into an MS culture medium added with 4.0mg/L6-BA and 0.5mg/LNAA to perform differential culture, wherein the pH value of the MS culture medium is controlled to be 6.4, the temperature of a culture room is 26 ℃, the illumination intensity is 1200lx, and the stem sophora flavescens sprouts are obtained after illumination is 14h and 39d every day;
4) Rooting and seedling strengthening culture: when the stem sophora flavescens differentiated bud in the step 3) grows into 4cm seedlings, transferring the stem sophora flavescens differentiated bud into an MS culture medium added with 1.0mg/LNAA and 1g/L activated carbon to carry out rooting and seedling strengthening culture, controlling the pH value of the MS culture medium to be 6.4, controlling the temperature of a culture room to be 26 ℃, the illumination intensity to be 1200lx, illuminating for 14h every day, and obtaining rooting seedlings after 49d;
5) Hardening and planting: culturing the rooted seedlings obtained in the step 4) in natural light for 3 days, opening the caps of the test tube seedlings, hardening off the seedlings for 3 days, lightly clamping the test tube seedlings by using forceps, washing off agar at the base, transplanting the test tube seedlings into sandy soil added with 1/2MS nutrient solution, transplanting the test tube seedlings into soil after 4 weeks, and performing field planting.
Example 11
A tissue culture method of radix sophorae flavescentis comprises the following steps:
1) And (3) explant sterilization: washing the vine and lightyellow sophora root fruit pods with sterile water; taking out the seeds in the fruit pods of the vine and lightyellow sophora root, soaking the seeds in 75% alcohol for 28s, and then washing the seeds with sterile water for 1 time; soaking the treated seeds with 0.1% mercuric chloride for 13min, and washing with sterile water for 5 times;
2) Seed germination culture: inoculating the seeds sterilized in the step 1) into an MS culture medium added with 1.0mg/L6-BA, 0.5mg/LNAA and 1g/L active carbon for germination culture, controlling the pH value of the MS culture medium to be 6.6, controlling the temperature of a culture room to be 26 ℃, the illumination intensity to be 1000lx, and obtaining sophora flavescens sprouts after illumination for 14h and 24d every day;
3) And (3) differentiated culture of cluster buds: inoculating the stem sophora flavescens sprouts obtained in the step 2) into an MS culture medium added with 3.0mg/L6-BA and 0.2mg/LNAA to perform differential culture, wherein the pH value of the MS culture medium is controlled to be 6.6, the temperature of a culture room is 26 ℃, the illumination intensity is 1000lx, and the stem sophora flavescens sprouts are obtained after illumination for 14h and 42d every day;
4) Rooting and seedling strengthening culture: when the stem sophora flavescens differentiated bud in the step 3) grows into 3-4cm seedlings, transferring the stem sophora flavescens differentiated bud into an MS culture medium added with 1.0mg/LNAA and 1g/L of activated carbon to carry out rooting and seedling strengthening culture, controlling the pH value of the MS culture medium to be 6.6, controlling the temperature of a culture room to be 26 ℃, controlling the illumination intensity to be 1000lx, illuminating for 14h every day, and obtaining rooted seedlings after 49d;
5) Hardening and planting: culturing the rooted seedlings obtained in the step 4) in natural light for 3 days, opening the bottle caps of the test tube seedlings, hardening the seedlings for 3 days, then slightly clamping the test tube seedlings by using forceps, washing off agar at the base part, transplanting the test tube seedlings into sandy soil added with 1/2MS nutrient solution, transplanting the test tube seedlings into soil after 4 weeks, and performing field planting.
Example 12
A tissue culture method of radix sophorae flavescentis comprises the following steps:
1) And (3) explant sterilization: washing the vine and lightyellow sophora root fruit pods with sterile water; taking out the seeds in the fruit pods of the sophora flavescens, soaking the seeds in 75% alcohol for 29s, and then washing the seeds with sterile water for 1 time; soaking the treated seeds with 0.1% mercuric chloride for 11min, and washing with sterile water for 4 times;
2) Seed germination culture: inoculating the seeds sterilized in the step 1) into an MS culture medium added with 1.5mg/L6-BA, 0.5mg/L LNAA and 1g/L active carbon for germination culture, controlling the pH value of the MS culture medium to be 6.5, controlling the temperature of a culture room to be 25 ℃, and the illumination intensity to be 1100lx, and obtaining the sophora flavescens sprouts after illumination for 12h and 20d every day, wherein the figure is 1;
3) And (3) differentiated culture of cluster buds: inoculating the stem lightyellow sophora root sprouts obtained in the step 2) into an MS culture medium added with 3.0mg/L6-BA and 0.5mg/LNAA to perform differentiation culture, wherein the pH value of the MS culture medium is controlled to be 6.5, the temperature of a culture room is 25 ℃, the illumination intensity is 1100lx, and the stem lightyellow sophora root differentiated sprouts are obtained after illumination for 14h and 35d every day, as shown in figure 2;
4) Rooting and seedling strengthening culture: when the stem sophora flavescens differentiated bud in the step 3) grows into 3-4cm seedlings, transferring the stem sophora flavescens differentiated bud into an MS culture medium added with 1.0mg/LNAA and 1g/L of activated carbon to carry out rooting and seedling strengthening culture, controlling the pH value of the MS culture medium to be 6.5, controlling the temperature of a culture room to be 25 ℃, the illumination intensity to be 1100lx, and obtaining rooted seedlings after 12h and 45d of illumination every day; as shown in FIG. 3;
5) Hardening and planting: culturing the rooted seedlings obtained in the step 4) in natural light for 3 days, opening the caps of the test tube seedlings, hardening off the seedlings for 3 days, lightly clamping the test tube seedlings by using forceps, washing off agar at the base, transplanting the test tube seedlings into sandy soil added with 1/2MS nutrient solution, transplanting the test tube seedlings into soil after 4 weeks, and performing field planting.
The tests were carried out as in examples 1 to 12, at the experimental sites: guangxi institute of traditional Chinese medicine tissue culture laboratory. Seed number: 30 granules. Counting the germination rate in the step 2), the number of the buds differentiated in the step 3), the condition of the rooted seedlings in the step 4) and the survival rate, wherein the counting is shown in a table 1;
TABLE 1
Example 1 Example 2 Example 3 Example 4 Example 5 Example 6 Example 7 Example 8 Example 9 Example 10 Example 11 Example 12
The germination rate% 80 80 50 80 90 80 60 70 95 80 100 100
Number of differentiated buds 31 30 30 40 40 35 58 40 26 26 31 58
Growth of shoots Strong bud Strong bud Strong bud Strong bud Strong bud Strong bud Strong bud Strong bud Strong bud Small and fine bud Strong bud Strong bud
Condition of rooted seedling Thick stem and root Thick stem and root Thick stem and root Thick stem and root Thick stem and root Thick stem and root Thick stem and root Thick stem and root Thick stem and root Thick stem and root Thick stem and root Thick stem and root
Survival rate 65.8 64.5 62.6 63.5 68.1 70.5 66.2 67.5 69.2 70.2 70.1 66.5
As can be seen from Table 1, in the seed germination culture, when the concentration of 6-BA is 1.0mg/L and 1.5mg/L, and the concentration of NAA is 0.5mg/L, the seed germination rate is 100%, and the bud growth is strong, so that in the actual production process, the use of 6-BA with a concentration of 1.0mg/L is more appropriate in consideration of the cost.
Example 13
100 litsea flavescens pods were tested in the manner described in example 12 and identified as control 1;
in the step 2) of example 12, 6-BA and NAA are not added and are marked as a control group 2;
in the embodiment 12, the concentration of 6-BA in the step 2) is 0.2mg/L, NAA is 0mg/L, and the result is marked as a test group 1;
in the embodiment 12, the concentration of 6-BA in the step 2) is 0.5mg/L, NAA is 0.2mg/L, and the result is marked as a test group 2;
in the embodiment 12, the concentration of 6-BA in the step 2) is 0.5mg/L, NAA is 1.0mg/L and is marked as a test group 3;
in the embodiment 12, the concentration of 6-BA in the step 2) is 0.8mg/L, NAA is 1.0mg/L, and the result is marked as a test group 4;
in the embodiment 12, the concentration of 6-BA in the step 2) is 1.0mg/L, NAA is 1.0mg/L and is marked as a test group 5;
in the step 2) of the example 12, the concentration of 6-BA is 1.5mg/L, NAA is 1.0mg/L and is marked as a test group 6;
in the embodiment 12, the concentration of 6-BA in the step 2) is 2.0mg/L, NAA is 1.0mg/L and is marked as a test group 7;
the germination rate and growth conditions in each group of this example were counted, see table 2;
TABLE 2
Control group 1 Control group 2 Test group 1 Test group 2 Test group 3 Test group 4 Test group 5 Test group 6 Test group 7
The germination rate% 100 50 50 60 40 40 60 70 60
Growth conditions Strong bud Small and fine bud Bud powder Small and fine bud Small and fine bud Bud powder Strong bud Strong bud Strong bud
Example 14
120 shoots were selected for inoculation and divided into 12 groups of 10 for each, and the test was performed:
the test was carried out in the manner of step 3) in example 12 and designated as control 1;
in the step 3) of the example 12, 6-BA and NAA are not added and are marked as a control group 2;
in the embodiment 12, the concentration of 6-BA in the step 3) is 0.5mg/L, NAA is 0.2mg/L, and the result is marked as a test group 1;
in the embodiment 12, the concentration of 6-BA in the step 2) is 1.0mg/L, NAA is 0.2mg/L, and the result is marked as a test group 2;
in the embodiment 12, the concentration of 6-BA in the step 2) is 2.0mg/L, NAA is 0.2mg/L, and the result is marked as a test group 3;
in the embodiment 12, the concentration of 6-BA in the step 2) is 5.0mg/L, NAA is 0.5mg/L, and the result is marked as a test group 4;
in the embodiment 12, the concentration of 6-BA in the step 2) is 0.5mg/L, NAA is 1.0mg/L and is marked as a test group 5;
in the step 2) of the example 12, the concentration of 6-BA is 1.0mg/L, NAA is 1.0mg/L and is marked as a test group 6;
in the embodiment 12, the concentration of 6-BA in the step 2) is 2.0mg/L, NAA is 1.0mg/L and is marked as a test group 7;
in the embodiment 12, the concentration of 6-BA in the step 2) is 3.0mg/L, NAA is 1.0mg/L, and the result is marked as a test group 8;
in the embodiment 12, the concentration of 6-BA in the step 2) is 4.0mg/L, NAA is 1.0mg/L, and the result is marked as a test group 9;
in the embodiment 12, the concentration of 6-BA in the step 2) is 5.0mg/L, NAA is 1.0mg/L and is marked as a test group 10;
the number of differentiated shoots and the growth potential of each group in this example were counted, as shown in table 3;
TABLE 3
Control group 1 Control group 2 Test group 1 Test group 2 Test group 3 Test group 4 Test group 5 Test ofGroup 6 Test group 7 Test group 8 Test group 9 Test group 10
Number of differentiated buds 58 10 12 10 22 13 10 10 12 12 9 10
Growth potential ++++ ++ +++ ++ +++ +++ ++++ ++++ +++ +++ ++ ++
Example 15
The test was carried out as in example 12 and identified as a control group;
in the case of rooting and seedling strengthening culture in example 12, activated carbon was not added and the test group was obtained;
counting the growth of the seedlings, see table 4;
TABLE 4
Control group Test group
Growth of the plantlet Thick stem and root The stem is slightly thin and the root is long
The embodiments in the present description are described in a progressive manner, each embodiment focuses on differences from other embodiments, and the same and similar parts among the embodiments are referred to each other. The device disclosed by the embodiment corresponds to the method disclosed by the embodiment, so that the description is simple, and the relevant points can be referred to the method part for description.
The previous description of the disclosed embodiments is provided to enable any person skilled in the art to make or use the present invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the generic principles defined herein may be applied to other embodiments without departing from the spirit or scope of the invention. Thus, the present invention is not intended to be limited to the embodiments shown herein but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims (3)

1. A tissue culture method of sophora flavescens ait is characterized by comprising the following steps:
1) And (3) explant sterilization: pretreating the fruit pods of the radix sophorae flavescentis, taking out seeds in the fruit pods, and disinfecting the seeds for later use;
the process of pretreating the fruit pods of radix sophorae flavescentis and disinfecting the seeds comprises the following steps:
11 Washing the fruit pod of radix Sophorae Flavescentis with sterile water for 3 times, each for 1-2 min;
12 Taking out the seeds in the fruit pods of the vine and lightyellow sophora root, soaking the seeds in 75% alcohol for 20-30s, and then washing the seeds with sterile water for 3 times, each time for 1-2 minutes;
13 Soaking the seeds treated in the step 12) for 10-15min by using 0.1% mercuric chloride, and then washing the seeds for 1min each time for 4-5 times by using sterile water for later use;
2) Seed germination culture: inoculating the seeds sterilized in the step 1) into an MS culture medium added with 0.8-2.0mg/L6-BA, 0.2-0.5mg/L NAA and 1g/L active carbon for germination culture, and obtaining vine sophora flavescens sprouts after 20-25 days;
3) And (3) differentiated culture of cluster buds: inoculating the stem sophora flavescens sprouts obtained in the step 2) into an MS culture medium added with 3.0-4.0mg/L6-BA and 0.2-0.5mg/L NAA for differential culture, and obtaining stem sophora flavescens differentiated sprouts after 35-45 days;
4) Rooting and seedling strengthening culture: when the stem sophora flavescens differentiated bud in the step 3) grows into 3-4cm seedlings, transferring the stem sophora flavescens differentiated bud into an MS culture medium added with 1.0mg/L NAA and 1g/L activated carbon to carry out rooting and seedling strengthening culture, and obtaining rooted seedlings after 45-55 days;
5) Hardening and planting: culturing the rooted seedlings obtained in the step 4) in natural light for 3 days, opening the bottle caps of the test tube seedlings, hardening the seedlings for 3 days, then slightly clamping the test tube seedlings by using forceps, washing off agar at the base part, transplanting the test tube seedlings into sandy soil added with 1/2MS nutrient solution, transplanting the test tube seedlings into soil after 4 weeks, and performing field planting.
2. The tissue culture method of Alopecurus rhamnoides according to claim 1, wherein in steps 2) -4), the temperature of the culture chamber is 23-27 ℃, the illumination intensity is 1000-1200lx, and the illumination time is 12-14 h/day.
3. The tissue culture method of Alstonia amurensis according to claim 1, wherein the pH value of the MS culture medium in the steps 2) -4) is controlled to 6.2-6.7.
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