CN115735771B - Efficient in-vitro propagation method for radix stemonae - Google Patents
Efficient in-vitro propagation method for radix stemonae Download PDFInfo
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Abstract
The invention relates to the technical field of rapid plant propagation, and particularly discloses a high-efficiency in-vitro propagation method of stemona. The method comprises the steps of (1) preparing an explant material; (2) fixed bud induction; (3) adventitious bud induction and proliferation; (4) adventitious bud elongation; and (5) rooting culture to obtain a complete plant. The method can efficiently reproduce the stemona in vitro, so that the resource waste of the seeds in the breeding process can be reduced, and excellent seedlings with stable characters and uniform quality can be stably provided for the market.
Description
Technical Field
The invention relates to the technical field of rapid plant propagation, and particularly discloses a high-efficiency in-vitro propagation method of stemona.
Background
Radix StemonaeStemona japonica(Blume) miq.), the genus stemona, an important Chinese medicinal material, has an application history of more than two thousand years in China. The root can be used as a medicine for external use for killing insects, relieving itching and killing lice; it has effects of moistening lung, relieving cough, and eliminating phlegm. Stemona root medicinal materials are completely collected and excavated wild resources, the natural reproductive capacity of Stemona root is weak, the growth is slow, the yield can be formed over 3 years, the demand is obviously greater than the yield, the medicinal materials are often out of stock and cut off, and the development of artificial planting is urgent.
The rare endangered plants are generally poor in self-reproduction capability in natural environment, the habitat is seriously interfered by human, the population quantity and distribution range are increasingly reduced, the protection and sustainable utilization of the rare endangered wild plants are one of the important research directions of the current protection biology, a large number of seedlings are urgently needed for on-site protection and natural regression to achieve the goal, the market demand of the rare medicinal plants is large, and the market demand of the conventional seedling production can not be met.
Stemona root is mainly propagated through seeds and root-divided propagation, and has the problems of low propagation coefficient, poor genetic stability, high production cost and the like. The efficient in-vitro regeneration technology is a reliable biotechnology for amplifying the population quantity of new excellent varieties and rare endangered species and realizing large-scale production of seedlings. However, the in vitro regeneration technology of the tree species is not mature, and has the technical difficulty: (1) difficulty in obtaining sterile materials; (2) low propagation coefficient; (3) lower content of medicinal effective components, etc.
Disclosure of Invention
Aiming at the actual needs and problems, the invention establishes a high-efficiency rapid propagation system of the stemona by the researches of aseptic system establishment, direct adventitious bud generation, adventitious root induction and plasma culture technology, takes tissue culture buds as a medicinal active ingredient accumulation carrier, quantitatively produces stemona buds rich in medicinal active ingredients, has short growth period and is not limited by seasons in raw material harvesting.
The invention provides a high-efficiency in-vitro propagation method of stemona, which comprises the following steps:
(1) Preparation of the explant material: taking 2-3 cm stemona branch as a starting explant and sterilizing;
(2) And (3) fixed bud induction: reserving a stem node of the sterile explant obtained in the step (1), and inoculating the stem node into a fixed bud induction culture medium of which the fixed bud induction culture medium is MS+0.1-0.5 mg/L NAA;
(3) Induction and proliferation of adventitious buds: inoculating the elongated fixed bud obtained in the step (2) to an adventitious bud induction culture medium by removing the old stake of the terminal bud zone, wherein the culture medium is MS+1-3 mg/L6-BA+0.1-1 mg/L ZT+0.1-0.5 mg/LNAA+0.5 g/L proline, and culturing for 8-10 weeks, preferably inoculating every 3-5 cycles;
(4) Adventitious bud elongation: transferring the adventitious bud cluster obtained in the step (3) into an adventitious bud elongation culture medium, wherein the elongation culture medium is MS+0.3-1.5 mg/L6-BA+0.03-0.15 mg/L NAA, and culturing 14-21 d;
(5) Rooting culture: inoculating the elongated cluster buds in the step (4) into a rooting culture medium, and culturing until rooting to obtain a complete plant;
sucrose 30/g/L, agar powder 5.5/g/L and pH value of the culture medium adjusted to 5.8 are added into the culture medium in the steps.
In specific implementation, the culture condition is 25+/-2 ℃, the illumination intensity is 2000-3000lx, and the illumination is 16 h/d.
Further, the method also comprises the step (6) of hardening off and transplanting: selecting the bottle seedlings which are rooted and strong in growth in the step (5), hardening the seedlings, and transplanting.
In a preferred embodiment, the sterilization in step (1) is specifically: treating with 5%potassium permanganate solution for 3-5 min, washing with clear water, placing the material into sterile triangular flask, transferring to ultra clean workbench, treating with 75% ethanol solution for 30-60 s, treating with PB disinfectant containing 50% plant tissue culture antibacterial agent PPM and 1% benzalkonium bromide mixed solution for 5-10 min, and rinsing with sterile water for 3-5 times.
In a further preferred embodiment, the fixed bud induction medium in step (2) is MS+0.3 mg/L NAA and germination begins upon inoculation into the axillary buds.
In a further preferred embodiment, the adventitious bud induction medium in step (3) is MS+ mg/L6-BA+0.1-0.5 mg/L ZT+0.3-0.5 mg/LNAA+0.5 g/L proline.
Still more preferably, the elongation medium in step (4) is MS+0.7 mg/L6-BA+0.03 mg/L NAA.
In one embodiment, the rooting medium in step (5) is 1/2 MS+0.1-1 mg/L IBA+1.5-2.5 mg/L NAA; preferably, the rooting medium in step (5) is 1/2MS+0.5 mg/L IBA+2 mg/L NAA.
In one specific embodiment, the seedling hardening method in the step (6) comprises the following steps: opening the culture bottle cap, injecting 0.5-1. 1 cm clear water, standing at room temperature under natural illumination for 2-3 days, picking out the bottle seedling, cleaning the culture medium attached to the base, and preparing for transplanting;
in another embodiment, the transplanting substrate transplanted in step (6) is peat soil: perlite: vermiculite = 3:1:1, immersing a substrate and a basin in water before transplanting to completely wet the substrate and basin, bagging and moisturizing after transplanting, spraying the surface of a plant by using a watering can when the humidity is insufficient, removing the bagging after the seedling grows strongly, and normally maintaining.
The invention determines the key links of efficient in vitro propagation of stemona through research and exploration, and obtains an efficient propagation method, thereby reducing the resource waste of seeds in the breeding process and stably providing excellent seedlings with stable characters and uniform quality for the market.
Drawings
Fig. 1 Stemona root tender shoot.
Fig. 2 Stemona sterile stem section.
Fig. 3 stemona root elongation fixed bud.
FIG. 4 adventitious bud induction phase I (30 d).
FIG. 5 adventitious bud induction phase II (70 d).
FIG. 6 adventitious bud elongation induction.
FIG. 7 Stemonae radix Confucius bud induced adventitious roots.
Fig. 8 Stemonae tissue culture seedlings.
Detailed Description
The invention is further illustrated by the following detailed description of specific embodiments, which is not intended to be limiting, but is made merely by way of example.
(1) Preparation of explant material
Taking 2-3 cm Stemona branch as initial explant (figure 1), treating with 5%potassium permanganate solution for 3-5 min, washing with clear water, placing the material into sterile triangular flask, transferring to ultra clean workbench, treating with 75% ethanol solution for 30-60 s, treating with PB (mixed solution of 50% plant tissue culture antiseptic PPM (Plant Preservative Mixture) and 1% benzalkonium bromide (Benzyldodecyldimethylammonium Bromide)) disinfectant for 3-12 min, and rinsing with sterile water for 3-5 times. The experimental results showed (table 1): the PB disinfectant has the advantages of short treatment time, high pollution rate, long treatment time and low pollution rate, but high inactivation rate; the PB disinfectant has better treatment time of 5-10 min, wherein the 75% ethanol is used for disinfecting for 40 s, the PB disinfectant has the best combined disinfection effect after being treated for 7 min, and the survival rate is 88.3% at most.
TABLE 1 Effect of different sterilization methods on Stemona explant viability
(2) Fixed bud induction
The obtained sterile explant, one stem node was kept, inoculated into fixed bud induction medium (FIG. 2) added with different cytokinin concentrations, and the medium was MS-based, and 0.1-0.5 mg/L NAA or 0.1-0.5 mg/L IBA was added. The experimental results showed (table 2): the bud induction effect of cytokinin NAA is superior to IBA, fixed buds can be effectively induced by adding 0.1-0.5 mg/L NAA, and the germination time is short, wherein the bud induction effect is optimal by adding 0.3 mg/L NAA (figure 3), the bud induction rate reaches 100%, and the axillary buds begin to sprout after about 3 d is inoculated.
TABLE 2 Effect of different auxin concentrations on fixed bud induction
(3) Adventitious bud induction and proliferation
The obtained elongation fixed bud (cutting off terminal bud) with the old stake is inoculated into a culture medium MS+1-3 mg/L6-BA+0.1-1 mg/L ZT+0.1-0.5 mg/LNAA+0.5 g/L proline, and is cultured for 60-70 d, and each 30 d transfer is carried out. The experimental results showed (table 3): the fixed buds with old piles do not differentiate into adventitious buds in an MS culture medium without hormone, and only the fixed buds are elongated. Different plant hormone combinations are added into an MS culture medium to be beneficial to the induction and proliferation of the adventitious buds of the stemona, the adventitious buds are induced to begin to differentiate about 14 and d, a certain number of adventitious buds are induced about 30 and d (figure 4), a large number of adventitious buds are induced by 60-70 d (figure 5), wherein the induction effect of the culture medium added with 2 mg/L6-BA, 0.1/0.5 mg/L ZT, 0.3/0.5 mg/LNAA and 0.5 g/L proline is optimal, the differentiation rate of the adventitious buds reaches 96.67%, and the average bud number reaches 28.67/explant.
(4) Elongation of adventitious bud
The induced adventitious bud cluster is transferred into adventitious bud elongation medium. The culture medium adopts MS as basic culture medium, 0.3-1.5 mg/L6-BA and 0.03-0.15 mg/L NAA are added, and 14-21 d are cultured. The results showed (table 4): the adventitious buds of Stemona sessilifolia can be induced to elongate by different combinations of plant hormones, but the induction effects of different combinations are different, the adventitious bud elongation effect is best when 0.7 mg/L6-BA and 0.03 mg/L NAA are added, and the adventitious bud elongation rate reaches 84.77% (figure 6).
(5) Rooting culture
The elongated plexus buds are inoculated into rooting medium. The culture medium uses 1/2MS as basic culture medium, and 0.1-1 mg/L IBA and 1.5-2.5 mg/L NAA are added. The results showed (table 5): the addition of cytokinin is favorable for the induction of adventitious roots of stemona cluster buds, 7-10 d adventitious roots start to sprout, and the addition of 0.5 mg/L IBA and 2 mg/L NAA has the best rooting induction effect (figure 7), the induction rate of the adventitious roots reaches 91.53%, and the average rooting number is 7.8.
(6) Hardening and transplanting
Selecting the root-rooted and strong stemona bottle seedlings for hardening off, wherein the hardening off method comprises the following steps: opening the culture bottle cap, injecting 0.5-1. 1 cm clear water, standing at room temperature under natural illumination for 2-3 days, taking out the seedling from the bottle, cleaning the culture medium attached to the base, and preparing for transplanting. The transplanting matrix is peat soil: perlite: vermiculite = 3:1:1, immersing the substrate and the basin in water before transplanting to completely wet the substrate and the basin, bagging the substrate and the basin after transplanting to preserve moisture, spraying the surface of the plant by using a watering can when the humidity is insufficient, removing the bagging after 10-14 and d, and normally maintaining the plant to grow strong (figure 8).
Claims (11)
1. Radix stemonaeStemona japonica(Blume) miq.) a high efficiency in vitro propagation method comprising the steps of:
(1) Preparation of the explant material: taking 2-3 cm stemona branch as a starting explant and sterilizing;
(2) And (3) fixed bud induction: reserving a stem node of the sterile explant obtained in the step (1), and inoculating the stem node into a fixed bud induction culture medium which is MS+0.1-0.5 mg/L NAA;
(3) Induction and proliferation of adventitious buds: inoculating the elongated fixed bud obtained in the step (2) to an adventitious bud induction culture medium by removing the old stake of the terminal bud zone, wherein the culture medium is MS+1-3 mg/L6-BA+0.1-1 mg/L ZT+0.1-0.5 mg/LNAA+0.5 g/L proline, and culturing for 8-10 weeks;
(4) Adventitious bud elongation: transferring the adventitious bud cluster obtained in the step (3) into an adventitious bud elongation culture medium, wherein the elongation culture medium is MS+0.3-1.5 mg/L6-BA+0.03-0.15 mg/L NAA, and culturing 14-21 d;
(5) Rooting culture: inoculating the elongated cluster buds in the step (4) into a rooting culture medium, and culturing until rooting to obtain a complete plant; the rooting culture medium is 1/2 MS+0.1-1 mg/L IBA+1.5-2.5 mg/L NAA
Sucrose 30/g/L, agar powder 5.5/g/L and pH value of the culture medium adjusted to 5.8 are added into the culture medium in the steps.
2. The efficient in vitro propagation method of stemona according to claim 1, wherein the culture condition is 25+/-2 ℃, the illumination intensity is 2000-3000lx and the illumination is 16 h/d.
3. The efficient in-vitro propagation method of stemona root of claim 1, further comprising the step of (6) hardening off and transplanting: selecting the bottle seedlings which are rooted and strong in growth in the step (5), hardening the seedlings, and transplanting.
4. The efficient in-vitro propagation method of stemona as claimed in claim 1, wherein the sterilization in the step (1) is specifically: treating with 5%potassium permanganate solution for 3-5 min, washing with clear water, placing the material into sterile triangular flask, transferring to ultra clean workbench, treating with 75% ethanol solution for 30-60 s, treating with PB disinfectant containing 50% plant tissue culture antibacterial agent PPM and 1% benzalkonium bromide mixed solution for 5-10 min, and rinsing with sterile water for 3-5 times.
5. The efficient in-vitro propagation method of stemona according to claim 1, wherein the fixed bud induction culture medium in the step (2) is MS+0.3 mg/L NAA, and the germination starts after the inoculation to the axillary buds.
6. The efficient in vitro propagation method of stemona according to claim 1, wherein the adventitious bud induction culture medium in the step (3) is MS+ mg/L6-BA+0.1-0.5 mg/L ZT+0.3-0.5 mg/LNAA+0.5 g/L proline.
7. The efficient in vitro propagation method of stemona according to claim 1, wherein every 3-5 cycles of cultivation in step (4) is inoculated.
8. The efficient in vitro propagation method of stemona according to claim 1, wherein the elongation medium in the step (4) is MS+0.7 mg/L6-BA+0.03 mg/L NAA.
9. The efficient in vitro propagation method of stemona according to claim 1, wherein the rooting medium in step (5) is 1/2ms+0.5 mg/L iba+2 mg/L NAA.
10. The efficient in-vitro propagation method of stemona root as claimed in claim 3, wherein the seedling hardening method in the step (6) is as follows: opening the culture bottle cap, injecting 0.5-1. 1 cm clear water, standing at room temperature under natural illumination for 2-3 days, picking out the bottle seedling, cleaning the culture medium attached to the base, and preparing for transplanting.
11. The efficient in-vitro propagation method of stemona root as claimed in claim 3, wherein the transplanting matrix transplanted in the step (6) is peat soil: perlite: vermiculite = 3:1:1, immersing a substrate and a basin in water before transplanting to completely wet the substrate and basin, bagging and moisturizing after transplanting, spraying the surface of a plant by using a watering can when the humidity is insufficient, removing the bagging after the seedling grows strongly, and normally maintaining.
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Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN104221873A (en) * | 2014-10-14 | 2014-12-24 | 南京帝道农业科技有限公司 | Fast reproduction method for sessiie siemona root tissue culture |
| CN106577291A (en) * | 2016-12-23 | 2017-04-26 | 广西中医药大学 | Method for callus efficient induction seedling development of stemona sessilifolia |
| CN114431153A (en) * | 2022-03-17 | 2022-05-06 | 西南林业大学 | Tissue culture rapid propagation method of medicinal plant stemona sessilifolia and application thereof |
| CN114467749A (en) * | 2022-01-21 | 2022-05-13 | 贵州民族大学 | Culture medium and method for rapid breeding of stemona tuberosa seedlings |
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Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN104221873A (en) * | 2014-10-14 | 2014-12-24 | 南京帝道农业科技有限公司 | Fast reproduction method for sessiie siemona root tissue culture |
| CN106577291A (en) * | 2016-12-23 | 2017-04-26 | 广西中医药大学 | Method for callus efficient induction seedling development of stemona sessilifolia |
| CN114467749A (en) * | 2022-01-21 | 2022-05-13 | 贵州民族大学 | Culture medium and method for rapid breeding of stemona tuberosa seedlings |
| CN114431153A (en) * | 2022-03-17 | 2022-05-06 | 西南林业大学 | Tissue culture rapid propagation method of medicinal plant stemona sessilifolia and application thereof |
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| "药用植物百部的组织培养与快速繁殖";杨振德等;《中草药》(第9期);第78-81页 * |
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