CN114885839B

CN114885839B - Method for rapidly propagating seedlings of stem of common andrographis

Info

Publication number: CN114885839B
Application number: CN202210533003.6A
Authority: CN
Inventors: 陈红锋; 张雯; 郑希龙; 陈国华
Original assignee: South China Botanical Garden of CAS
Current assignee: South China Botanical Garden of CAS
Priority date: 2022-05-11
Filing date: 2022-05-11
Publication date: 2022-12-30
Anticipated expiration: 2042-05-11
Also published as: CN114885839A

Abstract

The invention discloses a method for rapidly propagating seedlings of stem of common andrographis. A. Stem segments of Chuanxinteng as an explant, the plant is cultured in a culture medium, cleaning and disinfecting; B. and C, inoculating the explant sterilized in the step A into a culture medium for culture, wherein the culture medium is BA1-3mg/L + NAA0.05-0.2mg/L + agar + activated carbon + MS culture medium, and thus, a complete regeneration seedling with buds and roots is obtained. The method for rapidly propagating the seedlings of the stem-leaf dioscorea nipponica is characterized in that the current-year stem segment of the stem-leaf dioscorea nipponica is used as a starting material, the stem-leaf dioscorea nipponica can be cultured to generate a regenerated plant of the stem-leaf dioscorea nipponica in one step, multiple culture mediums are not needed to induce the formation of adventitious buds and roots step by step, the seedling culturing period of the stem-leaf dioscorea nipponica is greatly shortened, the resource waste in the breeding process is reduced, excellent seedlings with stable characters and uniform quality can be stably provided for the market, and the method has profound significance for sustainable development of medicinal plants of Yao nationality and reduction of breeding cost.

Description

Method for rapidly propagating seedlings of stem of common andrographis

Technical Field

The invention belongs to the field of medicinal plant tissue culture, and particularly relates to a method for rapidly propagating seedlings of stem of common andrographis.

Background

The plant of the genus Thamnolia of the family Araceae is a plant of the genus Thamnolia of the family Araceae, and is a unique plant of China. The seeds are distributed in places such as Guangdong, southeast of Yunnan, south of Hunan and Guangxi in China, grow in valley and water-side dense forest in the area with the elevation of 1300 m, are few in quantity, and have no artificial introduction and cultivation report. It is the heart-through wind of 104 classical old-class herbs commonly used as Yao medicine, and has the effects of clearing heat and removing toxicity, dispelling wind and removing dampness. Yao nationality areas are usually used for treating gastritis, cholecystitis bone tuberculosis, scabies, cellulitis and the like, and have remarkable curative effect.

The Chuanxinteng has good medicinal value, but the propagation of the Chuanxinteng is limited under natural conditions, the regeneration speed of resources is slow, and the resources are exhausted due to long-term unrestrained mining. In order to better protect the wild source of the stem of oriental paperbush, artificial cultivation needs to be developed vigorously to ensure the sustainable utilization of the stem of oriental paperbush. At present, the isolated regeneration and artificial cultivation of the Chinese polyphylla has not been reported at home and abroad.

The main mode of seedling culture is tissue culture, and the adopted process generally comprises a plurality of steps: inducing adventitious buds → differentiating adventitious buds → breeding culture → strengthening seedlings and rooting. The process needs various culture mediums for bottle changing and switching for many times, which not only wastes manpower and material resources, but also increases the pollution rate of tissue culture seedlings, so that the production cost of the seedlings is high. How to reduce the production cost of the seedlings and shorten the culture period is a problem which needs to be solved urgently in production. The research optimizes the seedling culture technology, researches the seedling culture technology of the explant, aims to further simplify the seedling culture technology, reduces the seedling production cost and provides technical support for the production technology of the stem-piercing germchit.

Disclosure of Invention

In order to meet the medicinal requirements of the stem of common andrographis and reduce the waste of resources, the invention simplifies the in vitro rapid propagation system of the stem of common andrographis, provides a method for rapidly propagating seedlings of the stem of common andrographis, and obtains a regenerated plant with higher frequency.

The invention improves the traditional tissue culture process: firstly, the current annual stem section of the stem of the common andrographis herb is selected as an initial material, the material is easy to be used as the initial material of one-step seedling, secondly, the MS culture medium is improved to be used as a basal culture medium, the induction and rooting can be realized by one step (only one culture medium is needed) by matching with a proper hormone proportion.

The invention relates to a method for rapidly propagating seedlings by stem segments of a perforator, which comprises the following steps:

A. stem segments of Chuanxinteng as an explant, the plant is cultured in a culture medium, cleaning and disinfecting;

B. and B, inoculating the explant sterilized in the step A into a culture medium for culture, wherein the culture medium is BA1-3mg/L + NAA0.05-0.2mg/L + agar + activated carbon + MS culture medium, and thus, a complete regeneration seedling with buds and roots is obtained.

Preferably, the concentration of the activated carbon is 0.05 percent of activated carbon and 6.0g/L of agar.

Preferably, the MS culture medium is an improved MS culture medium, potassium nitrate in macroelements in the MS culture medium is changed to 1200mg/L, ammonium nitrate is changed to 1200mg/L, monopotassium phosphate is changed to 210mg/L, magnesium sulfate is changed to 130mg/L, calcium chloride is changed to 150mg/L, and other elements are not changed.

Preferably, the culture medium contains 2.0mg/L of 6-BA and 0.1-0.2mg/L of NAA.

Further preferably, the culture medium contains 2.0mg/L of 6-BA and 0.2mg/L of NAA.

Preferably, the cleaning of the explant comprises the steps of shearing the current-year branch of the plant of the stem of the byttneria paniculata, shearing off the leaf and reserving part of the leaf stalk, cleaning stem nodes with a soft brush, soaking in sterilizing solution for 10min, and washing with tap water for more than 30 min. .

Preferably, the sterilization is carried out by sequentially scrubbing the epidermis of the stem segments with cotton balls impregnated with 75% ethanol, then cutting the segmented stem segments into 2cm pieces, rinsing the cut pieces in sterile water on a clean bench, sterilizing the cut pieces with 4% NaClO solution for 30s, washing the cut pieces with sterile water for 4-5 times, and 0.1% HgCl ₂ Sterilizing the water solution for 10-13min, and washing with sterile water for 4-5 times.

Preferably, the culture conditions in step B are 24 + -2 deg.C, 1800-2000lx light and 12h/d.

Transferring the regenerated seedlings obtained in the step B, the culture medium and the culture bottle into a greenhouse for tolerant culture for 3-6 days, opening the cover of the culture bottle, injecting 10-20ml of sterile water or deionized water, and hardening the seedlings for 5-8 days.

Preferably, the regenerated plant after hardening off is transplanted into a matrix of peat soil and perlite, and the regenerated plant grows in a greenhouse under the natural illumination at the temperature of 28 +/-2 ℃ to become the regenerated plant of the stem of oriental stephania consistent with the naturally grown seedling. The peat soil and the perlite are mixed according to the proportion of 5.

The method for rapidly propagating the seedlings of the stem-leaf dioscorea nipponica is characterized in that the current-year stem segment of the stem-leaf dioscorea nipponica is used as a starting material, the stem-leaf dioscorea nipponica can be cultured to generate a regenerated plant of the stem-leaf dioscorea nipponica in one step, multiple culture mediums are not needed to induce the formation of adventitious buds and roots step by step, the seedling culturing period of the stem-leaf dioscorea nipponica is greatly shortened, the resource waste in the breeding process is reduced, excellent seedlings with stable characters and uniform quality can be stably provided for the market, and the method has profound significance for sustainable development of medicinal plants of Yao nationality and reduction of breeding cost.

Description of the drawings:

FIG. 1 shows adventitious buds of multiple shoots induced at the stem node of a stem segment;

FIG. 2 shows the hormone ratios: 2.0mg/L of 6-BA and 0.2mg/L of NAA;

FIG. 3 shows the root system of the stem of Ningpo Yam after induction.

Detailed Description

The following examples further illustrate the invention, but are only given by way of illustration to clearly illustrate the invention and should not be construed as limiting the invention. Other variations and modifications will be apparent to persons skilled in the art in light of the above description. And are neither required nor exhaustive of all embodiments. And it is expressly intended that all such modifications or alterations to the methods, processes or conditions of the present invention as come within the scope of the invention are desired to be protected.

Example 1:

1. taking materials and cleaning: cutting off leaves of annual branches of caulis seu folium Stauntoniae collected from outdoors in sunny days, removing leaves and leaves of the annual branches, washing off soil, brushing stem nodes with a soft brush, mixing 2g of Guoguang carbendazim in 1L of water, soaking the branches for 10min, and washing with running water for more than 30min for later use;

2. and (3) disinfection: sequentially scrubbing the epidermis of stem segment with cotton ball soaked with 75% ethanol aqueous solution, cutting the stem segment into 2cm, washing in sterile water on a clean bench, sterilizing with 4% NaClO solution for 30s, washing with sterile water for 4-5 times, and processing into powder with 0.1% HgCl ₂ Sterilizing the water solution for 10-13min, and washing with sterile water for 4-5 times;

3. cutting off two ends of the stem of the sterilized stem of the dioscorea nipponica in the step 2, respectively inoculating the stem of the dioscorea nipponica in two basic culture media MS and improved MS which are added with 6-BA2.0mg/L, sucrose 25g/L and activated carbon with the mass fraction of 0.05 percent and agar 6.0g/L, carrying out single-factor analysis, and selecting the most suitable basic culture medium;

the improved MS culture medium is characterized in that potassium nitrate in macroelements in the MS culture medium is changed to 1200mg/L, ammonium nitrate is changed to 1200mg/L, monopotassium phosphate is changed to 210mg/L, magnesium sulfate is changed to 130mg/L, calcium chloride is changed to 150mg/L, and other elements are not changed.

The MS culture medium is a culture medium commonly used for plant tissue culture, the preparation method is common knowledge, the improved MS culture medium of the invention is prepared by changing potassium nitrate in a large amount of elements in the MS culture medium to 1200mg/L, ammonium nitrate to 1200mg/L, monopotassium phosphate to 210mg/L, magnesium sulfate to 130mg/L, calcium chloride to 150mg/L, and other elements are not changed, and the preparation method is the same as that of the MS culture medium.

Then adding 2.0mg/L of 6-BA, 25g/L of sucrose, 0.05 percent of activated carbon mass fraction and 6.0g/L of agar into an MS culture medium or an improved MS culture medium, and sterilizing at 121 ℃ for 15-25 minutes to obtain the product.

4. Subjecting the product obtained in step (2) cutting off two ends of stem of caulis Fibraureae, respectively inoculating on modified MS culture medium (also added with sucrose 25g/L + active carbon mass fraction 0.05% + agar 6.0 g/L) added with BA (1.0, 2.0, 3.0 mg/L) and NAA (0.05, 0.1, 0.2 mg/L) in different concentration ratio combinations (see Table 2 specifically). 10 stem segments were inoculated for each treatment, with 3 replicates. After 2-4 weeks of culture, new buds were induced at the stem node part of the stem segment (FIG. 1), at which time no root line of the base of Chuanxinteng occurred.

5. The culture was continued for 4-8 weeks with formation of roots (FIGS. 2 and 3).

The culture conditions of the steps 3, 4 and 5 are as follows: culturing under illumination of 1800lx for 12h/d at 24 + -2 deg.C;

transferring the rooted seedling obtained in the step 5, the culture medium and the culture bottle into a greenhouse for tolerant culture for 3-6 days, opening the cover of the culture bottle, injecting 10-20ml of sterile water or deionized water, and hardening the seedling for 5-8 days; then transplanting the seedlings into a mixed matrix of peat soil and perlite (5 v/v), and growing well in a greenhouse at 28 +/-2 ℃ under natural illumination to obtain the regenerated plants of the stem of the Periploca forrestii Turcz consistent with naturally grown seedlings;

among them, group 6 rooted better. And after culturing for 90 days, counting the germination rate and the one-step seedling rate.

The experimental results are as follows: from the perspective of bud induction rate and bud pollution rate, the bud induction rate in the improved MS culture medium is higher than that of the MS culture medium, and the pollution rate is lower than that of the MS culture medium; from the growth state of the buds, the buds induced by the improved MS culture medium are strong and have better growth vigor (Table 1). Wherein, the rooting rate (%) is calculated as the number of explants inducing adventitious roots/the total number of explants inoculated x 100%. The result shows that the rooting rate can reach 100 percent at most, the formula is added with 6-BA2.0mg/L and NAA0.2mg/L, the regenerated plant grows robustly, and the transplanting survival rate is high. 2.0mg/L of 6-BA and 0.1mg/L of NAA are added to have the secondary effect of adventitious root induction, and the rooting rate is 84.6 percent. The overall data show that the whole one-step seedling induction effect is good, and the induction period is 70-90 days (see table 2).

TABLE 1 Effect of different basic media on shoot growth

TABLE 2 Effect of different hormone ratios on one-step seedling Induction

The experiment shows that the current-year stem is used as an initial explant, hormones BA and NAA are added into an improved MS culture medium, and the concentration of the phytohormones is adjusted, so that the method for tissue culture one-step seedling of the kadsura longipedunculata is realized, wherein the budding rate and the rooting quantity are improved, the adventitious bud can be grown in one step in about 70-90d, the one-step seedling induction effect is good, the bud green is good, the adventitious root grows strongly, and the transplanting is easy to survive, wherein the optimal proportion of the hormones in the culture is as follows: 2.0mg/L of 6-BA, 0.2mg/L of NAA, 25g/L of cane sugar, 0.05 percent of active carbon mass fraction, 6.0g/L of agar and improved MS.

Claims

1. A method for rapidly propagating seedlings by stem segments of a punching rattan is characterized by comprising the following steps:

B. inoculating the explant sterilized in the step A into a culture medium for culture, wherein the culture medium is 6-BA2.0mg/L + NAA0.2mg/L + sucrose 25g/L + activated carbon mass fraction 0.05% + agar 6.0g/L + improved MS, and thus obtaining a complete regenerated seedling with buds and roots;

2. The method of claim 1, wherein the explant is cleaned by cutting the current year shoots of a plant of Ficus simplicissima lour, cutting off leaves and part of petiole, cleaning stem joints with soft brush, soaking in sterilizing solution for 10min, and washing with tap water flow for more than 30 min.

3. The method of claim 1, wherein the sterilizing is accomplished by sequentially scrubbing the epidermis of the stem segments with cotton balls stained with 75% ethanol, then cutting the segmented stem segments into 2cm pieces, rinsing the cut pieces in sterile water on a clean bench, sterilizing with 4% NaClO solution for 30s, rinsing 4-5 times with sterile water, 0.1% ₂ Sterilizing the water solution for 10-13min, and washing with sterile water for 4-5 times.

4. The method according to claim 1, wherein the culturing in step B is carried out under conditions of 24. + -. 2 ℃ and 1800-2000lx light, 12h/d.

5. The method according to claim 1, wherein the regenerated plantlets obtained in step B are transferred into a greenhouse together with the culture medium and a culture bottle for tolerant culture for 3-6 days, then the cover of the culture bottle is opened, 10-20ml of sterile water or deionized water is injected, and the plantlets are hardened for 5-8 days.

6. The method as claimed in claim 5, wherein the regenerated plant after acclimatization is transplanted into a matrix of peat soil and perlite, and grown in a greenhouse under natural illumination at 28 ± 2 ℃ to become a regenerated plant of Chuanxinteng which is consistent with the naturally grown plantlet.