CN115735771A

CN115735771A - High-efficiency in-vitro propagation method of radix stemonae

Info

Publication number: CN115735771A
Application number: CN202211525325.2A
Authority: CN
Inventors: 周江平; 周紫阳; 朱木兰; 郑珂瑗
Original assignee: Shanghai Bright Biotechnology Co ltd
Current assignee: Shanghai Bright Biotechnology Co ltd
Priority date: 2022-11-30
Filing date: 2022-11-30
Publication date: 2023-03-07
Anticipated expiration: 2042-11-30
Also published as: CN115735771B

Abstract

The invention relates to the technical field of plant rapid breeding, and particularly discloses a high-efficiency in-vitro breeding method of radix stemonae. The method comprises the steps of (1) preparing explant material; (2) bud setting induction; (3) adventitious bud induction and proliferation; (4) adventitious bud elongation; and (5) rooting culture to obtain a complete plant. The method can efficiently propagate the stemona in vitro, thereby reducing the resource waste of the seeds in the breeding process and stably providing excellent seedlings with stable characters and uniform quality for the market.

Description

High-efficiency in-vitro propagation method of radix stemonae

Technical Field

The invention relates to the technical field of plant rapid breeding, and particularly discloses a high-efficiency in-vitro breeding method of radix stemonae.

Background

Stemona root (Stemona root)Stemona japonica(Blume) miq.), important Chinese medicinal materials of stemonaceae stemona, have application history for two thousand years in China. The root can be used as a medicine and is externally used for killing insects, relieving itching and killing lice; it has the actions of moistening lung, relieving cough and eliminating phlegm. The radix stemonae medicinal material is completely dependent on digging wild resources, the radix stemonae has weak natural reproduction capability and slow growth, the yield can be formed for more than 3 years, the demand is obviously greater than the yield, the medicinal material is often out of stock and broken, and the development of artificial planting is urgent.

Rare or endangered plants generally have poor self-propagation capacity in natural environment, the habitat of the rare or endangered plants is seriously interfered by human, the number of habitats and the distribution range are increasingly reduced, the protection and sustainable utilization of the rare or endangered wild plants are one of the key research directions for protecting biology at present, a large number of seedlings are urgently needed for ex-situ protection and natural regression to fulfill the aim, the market demand of rare medicinal plants is large, and the market demand cannot be met by conventional seedling production.

Stemona tuberosa is mainly propagated by seeds and roots, 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 population quantity amplification of new and excellent varieties and rare endangered species and large-scale production of seedlings thereof. However, the in vitro regeneration technology of the tree species is not mature, and the technical difficulty is as follows: (1) difficulty in obtaining sterile materials; (2) the propagation coefficient is low; and (3) the content of the medicinal effective components is low, and the like.

Disclosure of Invention

Aiming at the practical needs and problems, the invention establishes a high-efficiency rapid propagation system of the stemona root by the research of in vitro culture technologies such as sterile system establishment, direct adventitious bud generation, adventitious root induction and the like, takes the tissue culture bud seedling as a medicinal effective component accumulation carrier, quantitatively produces the stemona root bud seedling rich in medicinal effective components, has short growth period and is not limited by seasons for raw material harvesting.

The invention provides a high-efficiency in vitro propagation method of radix stemonae, which comprises the following steps:

(1) Preparation of explant material: taking 2-3 cm stemona tuberosa branches as initial explants and sterilizing;

(2) And (3) bud setting induction: reserving a stem node of the sterile explant obtained in the step (1), and inoculating the stem node in a fixed bud induction culture medium which is MS +0.1-0.5 mg/L NAA;

(3) Adventitious bud induction and proliferation: cutting the extended fixed bud obtained in the step (2), cutting off the terminal bud with an old pile, inoculating the cut terminal bud with an old pile to an adventitious bud induction culture medium which 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, once every 3-5 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 cluster buds elongated in the step (4) into a rooting culture medium, and culturing until the cluster buds grow roots to obtain a complete plant;

in the steps, 30 g/L of sucrose and 5.5 g/L of agar powder are added into the culture medium, and the pH value of the culture medium is adjusted to 5.8.

In specific implementation, the culture conditions are 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: and (5) selecting bottle seedlings which have rooted and have strong growth in the step (5), hardening off the seedlings, and transplanting the seedlings.

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 in 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 another preferred embodiment, the adventitious bud induction medium in step (2) is MS +0.3 mg/L NAA, and sprouting begins when the axillary buds are inoculated.

In a further preferred embodiment, the adventitious bud induction medium in step (3) is MS +2 mg/L6-BA +0.1-0.5 mg/L ZT +0.3-0.5 mg/LNAA +0.5 g/L proline.

Even 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 1/2MS +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 a specific embodiment, the seedling exercising method in the step (6) is as follows: opening the culture bottle cap, injecting 0.5-1 cm clear water, standing for 2-3 days at room temperature under natural illumination, taking out the bottle seedling, cleaning the culture medium attached to the base part, and preparing for transplanting;

in another embodiment, the transplanting substrate transplanted in the step (6) is peat soil: perlite: vermiculite =3:1:1, soaking the matrix in water to completely wet the matrix before transplanting, bagging and moisturizing after transplanting, spraying the water onto the surface of a plant by using a spray can when the humidity is insufficient, removing the bag after the seedling grows sturdy, and normally maintaining.

According to the method, through research and exploration, the key link of efficient in-vitro breeding of the stemona is determined, and the efficient breeding method is obtained, so that the resource waste of the breeding seeds can be reduced, and excellent seedlings with stable characters and uniform quality can be stably provided for the market.

Drawings

FIG. 1 shows young stemona shoots.

FIG. 2 sterile Stem segments of Stemona sessilifolia.

FIG. 3 the extended adventitious bud of Stemona.

FIG. 4 adventitious bud induction phase I (30 d).

FIG. 5 adventitious bud Induction stage II (70 d).

FIG. 6 adventitious bud elongation induction.

FIG. 7 adventitious roots induced by stemona tuberosa.

FIG. 8 Stemona tissue culture seedling.

Detailed Description

The invention is further illustrated by the following detailed description of specific embodiments, which are not intended to be limiting but are merely exemplary.

(1) Preparation of explant Material

Taking 2-3 cm Stemona tuberosa as initial explant (figure 1), treating with 5 ‰ potassium permanganate solution for 3-5 min, washing with clear water, placing the material in sterile triangular flask, transferring to ultra-clean bench, treating with 75% ethanol solution for 30-60 s, treating PB (Mixture of 50% Plant tissue antibacterial agent PPM (Plant Preservative texture) and 1% benzalkonium Bromide (Benzyldodecyldiminium Bromide) disinfectant) for 3-12 min, and finally rinsing with sterile water for 3-5 times. The experimental results show (table 1): the PB disinfectant has too short treatment time, high pollution rate, too long treatment time, low pollution rate and high inactivation rate; the PB disinfectant has a good effect after 5-10 min treatment, wherein the combined disinfection effect of 75% ethanol disinfection for 40 s and PB disinfectant treatment for 7 min is the best, and the survival rate is 88.3% at most.

TABLE 1 Effect of different Disinfection methods on Stemona Stemonae explant viability

(2) Adventitious bud induction

The obtained sterile explants were kept on one stem node and inoculated into adventitious bud induction medium (FIG. 2) supplemented with different auxin concentrations using MS as minimal medium supplemented with 0.1-0.5 mg/L NAA or 0.1-0.5 mg/L IBA. The experimental results show (table 2): the cell growth hormone NAA has a bud induction effect superior to IBA, can effectively induce fixed buds by adding 0.1-0.5-mg/L NAA, has short germination time, has the best induction effect by adding 0.3 mg/L NAA (figure 3), has the bud induction rate of 100 percent, and can sprout when axillary buds are inoculated to about 3 d.

TABLE 2 Effect of different cell auxin concentrations on adventitious bud Induction

(3) Adventitious bud induction and proliferation

The obtained elongated fixed bud (the terminal bud is cut off) with the old pile 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 60-70 d is cultured, and the inoculation is carried out once per 30 d. The experimental results show (table 3): the adventitious bud with the old stump is not differentiated in the MS culture medium without adding hormone, and only the adventitious bud is elongated. Different phytohormone combinations are added into an MS culture medium to be beneficial to induction and proliferation of stemona adventitious buds, the induction of differentiation of adventitious buds about 14 d, the induction of a certain number of adventitious buds about 30 d (figure 4), the induction of a large number of adventitious buds about 60-70 d (figure 5), wherein the induction effect of the culture medium added with 2 mg/L6-BA, 0.1/0.5 mg/ZT L, 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 number of buds reaches 28.67 per explant.

(4) Elongation of adventitious bud

The induced adventitious bud plexus was transferred to adventitious bud elongation medium. MS is used as a basic culture medium, 0.3-1.5 mg/L6-BA and 0.03-0.15 mg/L NAA are added, and 14-21 d is cultured. The results show (table 4): different phytohormone combinations can induce adventitious bud elongation of the stemona, but different combinations have different inducing effects, the adventitious bud elongation effect is the best when the combination of 0.7 mg/L6-BA and 0.03 mg/L NAA is added, and the adventitious bud elongation rate reaches 84.77 percent (figure 6).

(5) Rooting culture

The elongated clumpy buds are inoculated into a rooting medium. The culture medium uses 1/2MS as a basic culture medium, and 0.1-1 mg/L IBA and 1.5-2.5 mg/L NAA are added. The results show (table 5): the addition of the auxin is beneficial to the induction of adventitious roots of the stemona tuberosa cluster buds, the germination of 7-10 d adventitious roots begins, the rooting induction effect is the best by adding 0.5 mg/L IBA and 2 mg/L NAA (figure 7), the induction rate of the adventitious roots reaches 91.53 percent, and the average number of the roots is 7.8.

(6) Hardening and transplanting seedlings

Selecting bottle seedlings of radix stemonae which have rooted roots and are strong in growth for hardening seedlings, wherein the seedling hardening method comprises the following steps: opening the culture bottle cap, injecting 0.5-1 cm clear water, standing at room temperature under natural light for 2-3 days, taking out the seedling from the bottle, cleaning the culture medium attached to the base part, and preparing for transplanting. The transplanting matrix is peat soil: perlite: vermiculite =3:1:1, soaking the substrate in water before transplanting to completely wet the substrate, bagging and moisturizing after transplanting, spraying the substrate on the surface of a plant by using a spray can when the humidity is insufficient, removing the bag after 10-14 d, normally maintaining, and enabling the plant to grow robustly (figure 8).

Claims

1. A high-efficiency in vitro propagation method of radix stemonae comprises the following steps:

2. The method for efficiently propagating stemona sessilifolia in vitro as claimed in claim 1, wherein the culture conditions are 25 +/-2 ℃, the illumination intensity is 2000-3000lx, and the illumination is 16 h/d.

3. The method for efficiently propagating stemona sessilifolia in vitro according to claim 1, further comprising the following steps (6) of hardening off and transplanting: and (5) selecting bottle seedlings which have rooted and have strong growth in the step (5), hardening off the seedlings, and transplanting the seedlings.

4. The method for propagating stemona sessilifolia in vitro as claimed in claim 1, wherein the disinfection in step (1) is specifically: treating with 5 ‰ potassium permanganate solution for 3-5 min, washing with clear water, placing the material in 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 sessilifolia as claimed in claim 1, wherein the adventitious bud induction culture medium in step (2) is MS +0.3 mg/L NAA, and germination begins when the culture medium is inoculated to axillary buds.

6. The stemona efficient in vitro propagation method as claimed in claim 1, wherein the adventitious bud induction culture medium in the step (3) is MS +2 mg/L6-BA +0.1-0.5 mg/L ZT +0.3-0.5 mg/LNAA +0.5 g/L proline.

7. The method for breeding stemona sessilifolia in vitro as claimed in claim 1, wherein the elongation culture medium in the step (4) is MS +0.7 mg/L6-BA +0.03 mg/L NAA.

8. The method for efficient in-vitro propagation of radix stemonae as claimed in claim 1, wherein the rooting medium in step (5) is 1/2MS +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.

9. The method for breeding stemona sessilifolia in vitro as claimed in claim 1, wherein the seedling exercising method in step (6) comprises the following steps: opening the culture bottle cap, injecting 0.5-1 cm clear water, standing at room temperature under natural illumination for 2-3 days, taking out the bottle seedling, cleaning the culture medium attached to the base part, and preparing for transplanting.

10. The method for high-efficiency in-vitro propagation of stemona as claimed in claim 1, wherein the transplanting matrix transplanted in the step (6) is peat soil: perlite: vermiculite =3:1:1, soaking the matrix in water to completely wet the matrix before transplanting, bagging and moisturizing after transplanting, spraying the surface of the plant by using a spray can when the humidity is insufficient, removing the bag after the seedling grows sturdy, and maintaining normally.