Efficient induction method of leontopodium leontopodum adventitious roots

Abstract

The invention discloses an efficient induction method of leontopodium leontopodum adventitious roots, and belongs to the technical field of plant tissue culture. According to the efficient induction method for the leontopodium leontopodum adventitious roots, a leontopodium leontopodum sterile system and sterile seedlings are established, calluses are induced, rooting of tissue culture seedlings is promoted, browning is inhibited, and the adventitious roots extend; then the adventitious roots are induced through three ways, and the rooting induction rate of the leontopodium leontopodum adventitious roots is remarkably increased; the problems of leontopodium leontopodum resource shortage and large market demand are solved; a uniform, rapid and feasible culture is induced; the leontopodium leontopodum adventitious root induction method can be industrially produced, and sufficient raw materials are provided for extraction of leontopodium leontopodum flavone; by means of the method, large-scale culture of the leontopodium leontopodum adventitious roots is achieved, a large number of medicine and cosmetic raw materials can be obtained at low cost in a short time, and an integrated production system can be formed with the medicine industry and the cosmetic industry.

Description

Technical Field

[0001] This invention relates to the field of plant tissue culture technology, and more specifically to a highly efficient method for inducing adventitious roots of Edelweiss, which is particularly suitable for large-scale cultivation of Edelweiss and the targeted accumulation of secondary metabolites (flavonoids, leonuric acid, etc.). Background Technology

[0002] Edelweiss ( Leontopodium leontopodioides Edelweiss is a perennial herb belonging to the genus Edelweiss in the family Asteraceae. It has both medicinal and ecological value. It is rich in flavonoids, terpenoids, edelweiss acid and other secondary metabolites, and has clear antioxidant, anti-inflammatory, antibacterial and hepatoprotective biological activities. It has broad application prospects in the fields of pharmaceutical preparation research and development and high-end cosmetic raw material production.

[0003] However, there are rigid bottlenecks in the supply of edelweiss resources: firstly, it is naturally distributed in high-altitude and cold mountainous areas, with a growth cycle of 2-3 years and a natural reproduction rate of less than 20%; secondly, due to habitat destruction and over-harvesting, wild resources have been listed as rare and protected species, which is far from meeting the market demand with an annual growth rate of more than 15%.

[0004] To address the resource constraints, the scientific community has attempted various artificial propagation techniques, but all have significant limitations: ① Seed propagation: Seed germination rate is less than 30%, seedling transplant survival rate is less than 50%, and large-scale production is not feasible; ② Division propagation: The annual propagation coefficient of a single plant is only 2-3 plants, and the propagation cycle exceeds 6 months, making rapid propagation impossible; ③ Callus suspension culture: Product purity is less than 60%, browning rate exceeds 40% within 20 days of culture, and no adventitious root-specific induction system has been formed—adventitious roots, as carriers for the synthesis of secondary metabolites, have a synthesis capacity 3-5 times that of callus tissue, thus becoming the optimal technical direction.

[0005] Existing techniques for inducing adventitious roots in *Edendrobium nobile* suffer from three major drawbacks: ① Low induction rate (generally 50%-60%), with insufficient disinfection resulting in a contamination rate exceeding 30%, and excessive disinfection leading to a 40% loss of explant viability; ② Low growth efficiency, with a single plant gaining less than 0.5 g of fresh weight within a 20-day culture period, making mass harvesting impossible; ③ Low and unstable product content, with the dry weight content of the key component, leonuric acid, often below 0.8 mg / g (not meeting the minimum standard of 1.0 mg / g for medicinal extraction). In-depth analysis reveals that traditional methods rely solely on single hormone treatments (such as IBA), failing to systematically integrate the synergistic effects of "explant pretreatment - culture medium optimization - environmental regulation," resulting in unsatisfactory technical outcomes.

[0006] Therefore, providing an efficient method for inducing adventitious roots of Edelweiss is a problem that urgently needs to be solved by those skilled in the art. Summary of the Invention

[0007] Therefore, the application provides a high-efficiency induction method of S. przewalskii adventitious roots, and develops a S. przewalskii adventitious root induction system with low browning, high induction and high product, which has important scientific research value and economic significance for S. przewalskii resource protection and industrialization development.

[0008] In order to achieve the above object, the application adopts the following technical scheme: A high-efficiency induction method of S. przewalskii adventitious roots, comprising the following steps: Step 1: Construction of S. przewalskii sterile system Full-grain and undamaged mature seeds of S. przewalskii are selected and treated by the following disinfection procedures: ① 75 % ethanol is oscillated for 10-30 s; ② 0.1 % mercury chloride solution is oscillated for 5-10 min; ③ the residual disinfectant is removed by washing with sterile distilled water for 5-6 times, each time for 3 min; the disinfected seeds are inoculated into hormone-free MS solid medium and cultured in a culture box at a temperature of 23-25 ℃, a light intensity of 1500-2000 lx, and a light time of 12 h / d; the culture is carried out for 10-14 days to obtain sterile seedlings with a height of 3-5 cm and expanded leaves, and a stable sterile system is constructed; the MS solid medium is a basic medium containing 30 g / L sucrose and 6.5 g / L agar, and the pH is 5.8.

[0009] Step 2: Directional selection of explants and anti-browning pretreatment Stem segments or root segments are selected from the sterile seedlings as the explants (the induction efficiency of the stem segments is 2.3 times that of the leaves): 1.0-1.5 cm long stem segments are cut, 1-2 full axillary buds are reserved, the base aging tissues and excess leaves are removed, and only the top 1 small leaf is reserved; 1.0-1.5 cm long root segments are cut, and the base aging part is removed; the stem segments or root segments are immersed in a pretreatment liquid and treated at 25 ℃ in the dark for 1-4 hours; the pretreatment liquid is 1 / 2 MS liquid medium containing 0.1-0.3 mmol / L silver nitrate and 50-100 mg / L ascorbic acid; the silver nitrate inhibits ethylene synthesis, the ascorbic acid removes active oxygen, and the two together control the browning rate of the explants within 5 %.

[0010] Step 3: Preparation of high-efficiency induction medium A high-efficiency induction method of firebush adventitious roots is provided, wherein steps 2 and 3 are replaced by steps 6 and 7.

[0011] Step 4: Subsection induction culture The pretreated stem segments or root segments are inoculated into high-efficiency induction medium, 3-5 pieces per 100 mL triangular flask (to avoid overcrowding and competition), and subjected to subsection culture in the dark first and then in the light: ① dark culture stage: 22-24 ℃ dark culture for 3-5 days to promote the initiation of root primordia; ② light culture stage: transferred into an environment with light intensity of 1500-2000 lx, light time of 12 h / d, temperature of 23-25 ℃, and relative humidity of 60-70 %, and cultured for 25-35 days to induce the formation of adventitious roots.

[0012] Step 5: Proliferation culture and product accumulation The induced adventitious roots are cut, and the root tips with a length of 2-3 cm are reserved to improve the proliferation rate, inoculated into a proliferation culture medium, and placed in a shaking bed for culture: temperature of 23-25 ℃, light intensity of 1000-1500 lx, light time of 12 h / d, and rotation speed of 100-120 r / min (to ensure sufficient dissolved oxygen), and cultured for 20-30 days; the proliferation culture medium is a 1 / 2MS liquid culture medium containing IBA 0.5-0.8 mg / L + sucrose 30-40 g / L + hydrolyzed casein 100-200 mg / L. The fresh weight increment of the root system in liquid shaking bed culture is increased by more than 60 % compared with that in solid culture.

[0013] Further, a high-efficiency induction method of firebush adventitious roots is provided, wherein steps 2 and 3 are replaced by steps 6 and 7: Step 6: Induction of firebush callus The tissues of the aseptic seedlings are used as explants to induce callus, which is used as the starting material for induction of adventitious roots.

[0014] 1) Explants: the leaves, petioles or stem segments of the aseptic seedlings are used as explants.

[0015] 2) Induction medium: based on MS or B5 medium, adding auxin (for starting cell dedifferentiation), cytokinin (synergistic effect with auxin, regulating callus growth), sucrose (as carbon source and osmotic regulator) 30 g / L, agar (as solidifying agent) 6-8 g / L, pH adjusted to 5.6-5.8; the auxin is 1.0-2.0 mg / L 2,4-D or 0.5-1.5 mg / L NAA; the cytokinin is 0.1-0.5 mg / L 6-BA.

[0016] 3) Cultivation: inoculating explants on induction medium, cultivating at 25°C in dark conditions for 20-30 days until loose, light yellow or light green granular callus is formed; subculturing every 2-3 weeks, subculturing 2 times; the subculture medium is based on MS medium, adding 0.5-1.0 mg / L 2,4-D and 0.1-0.5 mg / L 6-BA; to expand and maintain the vitality of callus.

[0017] Step 7: High-efficiency induction culture Inoculating callus on high-efficiency induction medium, after 45 days of induction, callus-induced adventitious roots are obtained, which are used for segmented induction culture.

[0018] Further, the method is applied to improve the induction rate of Ledum palustre adventitious roots.

[0019] Further, the method is applied to improve the content of total flavonoids and Ledum palustre acid in Ledum palustre adventitious roots.

[0020] Through the above technical solutions, compared with the prior art, the present disclosure provides a high-efficiency induction method of Ledum palustre adventitious roots, which effectively resists browning by using composite pretreatment, synergistic effect of silver nitrate and ascorbic acid, browning rate is reduced from 35% to ≤5%, cell viability is increased by more than 40%, and a high induction rate foundation is built; the medium formula is optimized, the combination of “IBA+NAA+low concentration 6-BA+Pro” is created, the induction rate is more than 85% (traditional 50-60%), and the problem of low efficiency of single hormone is solved; segmented culture is performed to promote growth, dark culture is performed to promote root primordia, and light culture is performed to promote elongation, 30-day average root length ≥4 cm, and single plant fresh weight increment ≥0.8 g (traditional 2 times); the product is up to standard and stable, total flavonoid dry weight 9.5-13.8% (traditional 1.8%), liquid proliferation efficiency increased by more than 60%; suitable for large-scale production: step standardization, cycle 50-65 days / batch, can be connected to a fermenter, and economic and ecological values are considered.

[0021] This invention is of great practical significance for promoting the development of the Edelweiss industry, expanding its application in the fields of medicine and cosmetics, and constructing a breeding technology system for rare and endangered Chinese medicinal materials to achieve sustainable development and utilization. Attached Figure Description

[0022] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on the provided drawings without creative effort.

[0023] Figure 1 These are sterile seedlings of Edelweiss.

[0024] Figure 2 It is the adventitious root of Edelweiss.

[0025] Figure 3 It is the callus tissue of Edelweiss. Detailed Implementation

[0026] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0027] Unless otherwise specified, all raw materials and reagents used in this invention are from the conventional market.

[0028] The abbreviations involved are: IBA: indole-3-butyric acid; 6-BA: 6-benzylaminopurine; NAA: naphthaleneacetic acid; 2,4-D: 2,4-dichlorophenoxyacetic acid; Pro: proline.

[0029] Example 1 A highly efficient method for inducing adventitious roots in Edelweiss includes the following steps: 1) Construction of a sterile system: Select plump, undamaged mature seeds of Edelweiss. Disinfect the seeds by shaking with 75% ethanol for 30 seconds, then with 0.1% mercuric chloride solution for 5 minutes. Rinse with sterile water 5 times, 3 minutes each time. Inoculate the sterilized seeds onto hormone-free MS solid medium (MS as the basal medium, containing 30 g / L sucrose, 6.5 g / L agar, pH 5.8) and incubate at 24℃ with 1800 lx light for 12 h / d for 12 days to obtain sterile seedlings with a height of 3-5 cm and fully expanded leaves. Figure 1Then, the germination rate and contamination rate were measured (germination rate: 15 days after seed inoculation into the culture medium; contamination rate: 30 days after seed inoculation into the culture medium).

[0030] 2) Explant treatment: Cut 1.2 cm long stem segments from sterile seedlings, retain 1-2 plump axillary buds, remove aging tissue and excess leaves at the base, and retain only one small leaf at the top; immerse the stem segments in the pretreatment solution (1 / 2 MS liquid medium containing 0.2 mmol / L silver nitrate and 80 mg / L ascorbic acid) and treat in the dark at 25°C for 1 hour.

[0031] 3) High-efficiency induction medium: 1 / 2 MS + IBA 1.0 mg / L + NAA 0.3 mg / L + 6-BA 0.08 mg / L + sucrose 30 g / L + agar 6.5 g / L + Pro 80 mg / L, pH 5.8, autoclave at 121℃ for 20 min for later use.

[0032] 4) Induction Culture: Add 40 mL of high-efficiency induction medium to 100 mL Erlenmeyer flasks, inoculate 4 stem segments into each flask, and culture in the dark at 23 ℃ for 4 days. Then, transfer to an environment with 1800 lx light for 12 h / d, 24 ℃, and 65% humidity for 30 days to induce adventitious root formation. The adventitious root induction rate and average root length were measured (adventitious root induction rate: the percentage of explants that successfully induced adventitious roots 45 days after inoculation out of the total number of explants participating in the induction experiment; average root length: the average value obtained by dividing the sum of the lengths of all adventitious roots by the total number of adventitious roots).

[0033] 5) Proliferation Culture: Adventitious roots were cut, retaining 2-3 cm of root tip, and inoculated into proliferation medium (1 / 2 MS liquid medium + IBA 0.6 mg / L + sucrose 35 g / L + hydrolyzed casein 150 mg / L), and cultured at 24℃, 1200 lx light for 12 h / d, and 110 r / min for 25 days. After collecting, washing, drying, and grinding the adventitious roots of *Edelweiss*, the following indicators were determined: (Total flavonoid dry weight determination method: *Edelweiss* adventitious roots were dried and ground after 45 days of growth, and the determination was performed using the aluminum nitrate staining method and a UV spectrophotometer. *Edelweiss acid dry weight content: *Edelweiss* adventitious roots were dried and ground after 45 days of growth, and the determination was performed using high performance liquid chromatography).

[0034] Example 2 A highly efficient method for inducing adventitious roots in Edelweiss includes the following steps: 1) Construction of sterile system: Select plump and undamaged mature seeds of Edelweiss, shake with 75% ethanol for 15 s, shake with 0.1% mercuric chloride solution for 8 min for sterilization, rinse with sterile water 5 times for 3 min each time; inoculate the sterilized seeds into hormone-free MS solid medium (MS as the basal medium, containing 30 g / L sucrose, 6.5 g / L agar, pH 5.8), and incubate in an incubator: culture at 23℃, 1500 lx light for 12 h / d for 14 days to obtain sterile seedlings with a plant height of 3-5 cm and fully expanded leaves.

[0035] 2) Explant treatment: Cut 1.0 cm long stem segments from sterile seedlings, retain 1-2 plump axillary buds, remove aging tissue and excess leaves at the base, and retain only the top leaflet; immerse the stem segments in the pretreatment solution (1 / 2 MS liquid medium containing 0.1 mmol / L silver nitrate + 50 mg / L ascorbic acid) and treat in the dark at 25°C for 2 hours.

[0036] 3) High-efficiency induction medium: 1 / 2 MS + IBA 0.8 mg / L + NAA 0.2 mg / L + 6-BA 0.05 mg / L + sucrose 25 g / L + agar 6 g / L + Pro 50 mg / L, pH 5.7, autoclave at 121℃ for 20 min for later use.

[0037] 4) Induction culture: Add 40 mL of high-efficiency induction medium to 100 mL Erlenmeyer flasks, inoculate 3 stem segments into each flask, culture in the dark at 22℃ for 3 days, then transfer to 1500 lx light for 12 h / d, culture at 23℃ and 60% humidity for 25 days to induce adventitious root formation.

[0038] 5) Proliferation culture: Adventitious roots were cut, retaining 2-3 cm of root tip, and inoculated into proliferation medium (1 / 2 MS + IBA 0.5 mg / L + sucrose 30 g / L + hydrolyzed casein 100 mg / L), and cultured at 23 ℃, 1000 lx light for 12 h / d, and 100 r / min for 20 days. Figure 2 ).

[0039] Example 3 A highly efficient method for inducing adventitious roots in Edelweiss includes the following steps: 1) Construction of sterile system: Select plump and undamaged mature seeds of Edelweiss, sterilize them by shaking with 75% ethanol for 30s, then shake with 0.1% mercuric chloride solution for 10 min, and rinse with sterile water 5 times for 3 min each time; inoculate the sterilized seeds into hormone-free MS solid medium (MS as the basal medium, containing 30 g / L sucrose, 6.5 g / L agar, pH 5.8), and incubate them in an incubator: 25 ℃, 2000 lx light for 12 h / d for 10 days to obtain sterile seedlings with a height of 3-5 cm and fully expanded leaves.

[0040] 2) Explant treatment: Cut 1.5 cm stem segments from sterile seedlings, retain 1-2 plump axillary buds, remove aging tissue and excess leaves at the base, and retain only the top leaflet; immerse the stem segments in the pretreatment solution (1 / 2 MS liquid medium containing 0.3 mmol / L silver nitrate + 100 mg / L ascorbic acid) and treat in the dark at 25°C for 3 hours.

[0041] 3) High-efficiency induction medium: 1 / 2 MS + IBA 1.2 mg / L + NAA 0.4 mg / L + 6-BA 0.1 mg / L + sucrose 35 g / L + agar 7 g / L + Pro 100 mg / L, pH 5.9, autoclave at 121℃ for 20 min for later use.

[0042] 4) Induction culture: Add 40 mL of high-efficiency induction medium to 100 mL Erlenmeyer flasks, inoculate 5 stem segments into each flask, culture in the dark at 24℃ for 5 days, then transfer to 2000 lx light for 12 h / d, culture at 25℃ and 70% humidity for 35 days to induce adventitious root formation.

[0043] 5) Proliferation culture: Cut adventitious roots, retain 2-3 cm of root tip, and inoculate them into proliferation medium (1 / 2 MS + IBA 0.8 mg / L + sucrose 40 g / L + hydrolyzed casein 200 mg / L), and culture at 25℃, 1500 lx light for 12 h / d, and 120 r / min for 30 days.

[0044] Example 4 A highly efficient method for inducing adventitious roots in Edelweiss includes the following steps: 1) Construction of sterile system: Select plump and undamaged mature seeds of Edelweiss, shake with 75% ethanol for 15 seconds, shake with 0.1% mercuric chloride solution for 3 minutes for sterilization, and rinse with sterile water 6 times for 3 minutes each time; inoculate the sterilized seeds into hormone-free MS solid medium (MS as the basal medium, containing 30 g / L sucrose, 6.5 g / L agar, pH 5.8), and incubate in an incubator: 25 ℃, 2000 lx light for 12 h / d for 10 days to obtain sterile seedlings with a height of 3-5 cm and fully expanded leaves.

[0045] 2) Explant treatment: Cut 1.5 cm roots of Edelweiss from sterile seedlings and immerse them in pretreatment solution (1 / 2 MS liquid medium containing 0.3 mmol / L silver nitrate + 100 mg / L ascorbic acid) and treat in the dark at 25°C for 4 hours.

[0046] 3) High-efficiency induction medium: 1 / 2 MS + IBA 1.2 mg / L + NAA 0.4 mg / L + 6-BA 0.05 mg / L + sucrose 35 g / L + agar 7 g / L + Pro 50 mg / L, pH 5.8, autoclave at 121℃ for 20 min for later use.

[0047] 4) Induction culture: Add 40 mL of high-efficiency induction medium to 100 mL Erlenmeyer flasks, inoculate 5 roots in each flask, culture in the dark at 24 ℃ for 5 days, then transfer to 2000 lx light for 12 h / d, culture at 25 ℃ and 70% humidity for 35 days to induce adventitious root formation.

[0048] 5) Proliferation culture: Cut adventitious roots, retain 2-3 cm of root tip, and inoculate them into proliferation medium (1 / 2 MS + IBA 0.6 mg / L + sucrose 30 g / L + hydrolyzed casein 200 mg / L), and culture at 25 ℃, 1500 lx light for 12 h / d, and 120 r / min for 30 days.

[0049] Example 5 A highly efficient method for inducing adventitious roots in Edelweiss includes the following steps: 1) Construction of sterile system: Select plump and undamaged mature seeds of Edelweiss, shake with 75% ethanol for 10 seconds, shake with 0.1% mercuric chloride solution for 5 minutes for sterilization, and rinse with sterile water 6 times for 3 minutes each time; inoculate the sterilized seeds into hormone-free MS solid medium (MS as the basal medium, containing 30 g / L sucrose, 6.5 g / L agar, pH 5.8), and incubate in an incubator: 25 ℃, 2000 lx light for 12 h / d for 10 days to obtain sterile seedlings with a height of 3-5 cm and fully expanded leaves.

[0050] 2) Explant treatment: Cut 1.5 cm roots of Edelweiss from sterile seedlings and immerse them in pretreatment solution (1 / 2 MS liquid medium containing 0.3 mmol / L silver nitrate + 100 mg / L ascorbic acid) and treat in the dark at 25°C for 1 hour.

[0051] 3) High-efficiency induction medium: 1 / 2 MS + IBA 1.2 mg / L + NAA 0.4 mg / L + 6-BA 0.1 mg / L + sucrose 35 g / L + agar 7 g / L + Pro 100 mg / L, pH 5.8, autoclave at 121℃ for 20 min for later use.

[0052] 4) Induction culture: Add 40 mL of high-efficiency induction medium to 100 mL Erlenmeyer flasks, inoculate 5 roots in each flask, culture in the dark at 24 ℃ for 5 days, then transfer to 2000 lx light for 12 h / d, culture at 25 ℃ and 70% humidity for 35 days to induce adventitious root formation.

[0053] 5) Proliferation culture: Cut adventitious roots, retain 2-3 cm of root tip, and inoculate them into proliferation medium (1 / 2 MS + IBA 0.8 mg / L + sucrose 40 g / L + hydrolyzed casein 200 mg / L), and culture at 25℃, 1500 lx light for 12 h / d, and 120 r / min for 30 days.

[0054] Example 6 A highly efficient method for inducing adventitious roots in Edelweiss includes the following steps: 1) Construction of sterile system: Select plump and undamaged mature seeds of Edelweiss, shake with 75% ethanol for 10s, shake with 0.1% mercuric chloride solution at 120 r / min for 5 min for sterilization, rinse with sterile water 6 times, 3 min each time; inoculate the sterilized seeds into hormone-free MS solid medium (MS as the basal medium, containing 30 g / L sucrose, 6.5 g / L agar, pH 5.8), and incubate in an incubator: 25 ℃, 2000 lx light for 12 h / d for 10 days to obtain sterile seedlings with a height of 3-5 cm and fully expanded leaves.

[0055] 2) Callus induction: 0.5 cm × 0.5 cm pieces of aseptic seedling leaves were cut and inoculated into induction medium (MS + 1.2 mg / L 2,4-D + 0.5 mg / L 6-BA + 7 g / L agar, pH 5.8, 30 g / L sucrose), and cultured in the dark at 25 ℃ for 28 days to obtain loose, pale yellow callus tissue (induction rate 96%). Subculture was performed every 3 weeks using subculture medium (MS + 0.8 mg / L 2,4-D + 0.3 mg / L 6-BA), repeating twice. Figure 3 ).

[0056] 3) High-efficiency induction medium: The callus tissue was inoculated onto high-efficiency induction medium, 1 / 2 MS + IBA 1.0 mg / L + NAA 0.3 mg / L + 6-BA 0.1 mg / L + sucrose 30 g / L + agar 6 g / L + Pro 80 mg / L; pH 5.8, autoclaved at 121℃ for 20 min and set aside. After 45 days of induction, the adventitious root induction of the callus tissue was observed.

[0057] 4) Induction culture: Add 40 mL of high-efficiency induction medium to 100 mL Erlenmeyer flasks, inoculate 5 pieces of callus tissue to induce adventitious roots in each flask, culture in the dark at 24 ℃ for 3 days, then transfer to 2000 lx light for 12 h / d, culture at 25 ℃ and 70% humidity for 35 days to induce adventitious root formation.

[0058] 5) Proliferation culture: Cut adventitious roots, retain 2-3 cm of root tip, and inoculate them into proliferation medium (1 / 2 MS + IBA 0.7 mg / L + sucrose 35 g / L + hydrolyzed casein 200 mg / L), and culture at 25 ℃, 1500 lx light for 12 h / d, and 120 r / min for 30 days.

[0059] Comparative Example 1 (excluding pretreatment steps) 1) Construction of sterile system: Select plump and undamaged mature seeds of Edelweiss, shake with 75% ethanol for 15 s, shake with 0.1% mercuric chloride solution for 8 min for sterilization, rinse with sterile water 5 times for 3 min each time; inoculate the sterilized seeds into hormone-free MS solid medium (MS as the basal medium, containing 30 g / L sucrose, 6.5 g / L agar, pH 5.8), and incubate in an incubator: culture at 23℃, 1500 lx light for 12 h / d for 14 days to obtain sterile seedlings with a height of 3-5 cm and fully expanded leaves.

[0060] 2) High-efficiency induction medium: 1 / 2 MS + IBA 0.8 mg / L + NAA 0.2 mg / L + 6-BA 0.05 mg / L + sucrose 25 g / L + agar 6 g / L + Pro 50 mg / L, pH 5.7, autoclave at 121℃ for 20 min for later use.

[0061] 3) Induction culture: Add 40 mL of high-efficiency induction medium to 100 mL Erlenmeyer flasks, inoculate 3 stem segments into each flask, culture in the dark at 22℃ for 3 days, then transfer to 1500 lx light for 12 h / d, culture at 23℃ and 60% humidity for 25 days to induce adventitious root formation.

[0062] 4) Proliferation culture: Cut adventitious roots, retain 2-3 cm of root tip, and inoculate them into proliferation medium (1 / 2 MS + IBA 0.5 mg / L + sucrose 30 g / L + hydrolyzed casein 100 mg / L), and culture at 23 ℃, 1000 lx light for 12 h / d, and 100 r / min for 20 days.

[0063] Comparative Example 2 (without using a high-efficiency induction medium) 1) Construction of sterile system: Select plump and undamaged mature seeds of Edelweiss, shake with 75% ethanol for 30s, sterilize with 0.1% mercuric chloride solution at 120 r / min for 5min, rinse with sterile water 5 times, 3min each time; inoculate the sterilized seeds into hormone-free MS solid medium (MS as the basal medium, containing 30 g / L sucrose, 6.5 g / L agar, pH 5.8), and incubate in an incubator: 24℃, 1800 lx light for 12h / d for 12 days to obtain sterile seedlings with a height of 3-5 cm and fully expanded leaves.

[0064] 2) Explant treatment: Cut 1.2 cm long stem segments from sterile seedlings, retain 1-2 plump axillary buds, remove aging tissue and excess leaves at the base, and retain only one small leaf at the top; immerse the stem segments in the pretreatment solution (1 / 2 MS liquid medium containing 0.2 mmol / L silver nitrate and 80 mg / L ascorbic acid) and treat in the dark at 25°C for 1 hour.

[0065] 3) Traditional induction medium: 1 / 2 MS + IBA 1.0 mg / L + sucrose 30 g / L + agar 6.5 g / L, pH 5.8, autoclave at 121℃ for 20 min for later use.

[0066] 4) Induction culture: Add 40 mL of induction culture medium to 100 mL Erlenmeyer flasks, inoculate 4 stem segments into each flask, culture in the dark at 23℃ for 4 days, then transfer to an environment with 1800 lx light for 12 h / d, 24 ℃, and 65% humidity for 30 days to induce adventitious root formation.

[0067] 5) Proliferation culture: Cut adventitious roots, retaining 2-3 cm of root tip, and inoculate them into proliferation medium (1 / 2 MS liquid medium + IBA 1.5 mg / L + NAA 0.5 mg / L + 6-BA 0.5 mg / L + sucrose 35 g / L + hydrolyzed casein 150 mg / L), and culture at 24℃, 1200 lx light for 12 h / d, and 110 r / min in a shaker for 25 days.

[0068] Comparative Example 3 1) Construction of sterile system: Select plump and undamaged mature seeds of Edelweiss, shake with 75% ethanol for 30s, shake with 0.1% mercuric chloride solution for 3min for sterilization, rinse with sterile water 5 times, 3min each time; inoculate the sterilized seeds into hormone-free MS solid medium (MS as the basal medium, containing 30 g / L sucrose, 6.5 g / L agar, pH 5.8), and incubate in an incubator: 24℃, 1800lx light for 12h / d for 12 days to obtain sterile seedlings with a height of 3-5 cm and fully expanded leaves.

[0069] 2) Explant treatment: Cut a 1.2 cm long stem segment from a sterile seedling, retain 1-2 plump axillary buds, remove the aging tissue and excess leaves at the base, and retain only the top leaflet.

[0070] 3) Induction medium: 1 / 2 MS + IBA 1.0 mg / L + sucrose 30 g / L + agar 6.5 g / L, pH 5.8, autoclave at 121℃ for 20 min for later use.

[0071] 4) Induction culture: Add 40 mL of induction culture medium to 100 mL Erlenmeyer flasks, inoculate 4 stem segments into each flask, culture in the dark at 23 ℃ for 4 days, then transfer to an environment with 1800 lx light for 12 h / d, 24 ℃ and 65% humidity for 30 days to induce adventitious root formation.

[0072] 5) Proliferation culture: Cut adventitious roots, retain 2-3 cm of root tip, and inoculate them into proliferation medium (1 / 2 MS liquid medium + IBA 1.0 mg / L + sucrose 35 g / L + hydrolyzed casein 150 mg / L), and culture at 24℃, 1200 lx light for 12 h / d, and 110 r / min in a shaker for 25 days.

[0073] Comparative Example 4 1) Construction of sterile system: Select plump and undamaged mature seeds of Edelweiss, shake with 75% ethanol for 30s, sterilize with 0.1% mercuric chloride solution at 120 r / min for 5min, rinse with sterile water 5 times, 3min each time; inoculate the sterilized seeds into hormone-free MS solid medium (MS as the basal medium, containing 30 g / L sucrose, 6.5 g / L agar, pH 5.8), and incubate in an incubator: 24℃, 1800 lx light for 12h / d for 12 days to obtain sterile seedlings with a height of 3-5 cm and fully expanded leaves.

[0074] 2) Explant treatment: Cut 1.2 cm roots from sterile seedlings.

[0075] 3) Induction medium: MS + IBA 1.0 mg / L + sucrose 30 g / L + agar 6.5 g / L, pH 5.8, autoclave at 121℃ for 20 min for later use.

[0076] 4) Induction culture: Add 40 mL of induction culture medium to 100 mL Erlenmeyer flasks, inoculate 4 root segments into each flask, culture in the dark at 23 ℃ for 4 days, then transfer to an environment with 1800 lx light for 12 h / d, 24 ℃ and 65% humidity for 30 days to induce adventitious root formation.

[0077] 5) Proliferation culture: Cut adventitious roots, retain 2-3 cm of root tip, and inoculate them into proliferation medium (1 / 2MS liquid medium + IBA 1.0 mg / L + sucrose 35 g / L + hydrolyzed casein 150 mg / L), and culture at 24℃, 1200 lx light for 12 h / d, and 110 r / min in a shaker for 25 days.

[0078] The effects of sterile Edelweiss seedling growth and adventitious root induction in Examples 1-6 and Comparative Examples 1-4 were compared, and the results are shown in Table 1.

[0079] Table 1. Comparison of the effects of different treatments on the growth and adventitious root induction of Edelweiss aseptic seedlings.

[0080] As shown in Table 1, in Example 5, the seed germination rate was highest (up to 90%) when seeds were disinfected by soaking in 75% ethanol for 10 seconds and then by disinfecting with 0.1% mercuric chloride for 5 minutes, while the contamination rate was also the lowest (10%). In adventitious root induction, the root had the highest induction rate, followed by the stem segment, and finally the callus tissue.

[0081] In Example 5, the 1 / 2 MS medium with 0.8 mg / L IBA, 40 g / L sucrose, and 200 mg / L hydrolyzed casein was the optimal medium for inducing adventitious roots, and the adventitious roots cultured from it had the highest contents of flavonoids and edelweiss.

[0082] in conclusion This invention, through the synergistic innovation of "pretreatment-culture medium-segmented culture", significantly outperforms traditional methods in terms of induction rate, growth rate, and product content. Moreover, the technical parameters can be standardized, making it a promising candidate for large-scale industrial application.

[0083] The above description of the disclosed embodiments enables those skilled in the art to make or use the invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the invention. Therefore, the invention is not 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

1. A highly efficient method for inducing adventitious roots in Edelweiss, characterized in that, Includes the following steps: Step 1: Construction of the Edelweiss Aseptic System Select plump, undamaged mature seeds of Edelweiss and treat them with the following disinfection process: ① Shake with 75% ethanol for 10-30 seconds; ② Transfer to 0.1% mercuric chloride solution and shake to sterilize for 5-10 min; ③ Rinse with sterile distilled water 5-6 times, 3 min each time; Inoculate the sterilized seeds into MS solid medium and incubate in an incubator: temperature 23-25 ​​℃, light intensity 1500-2000 lx, light duration 12 h / d, culture for 10-14 days to obtain sterile seedlings with a height of 3-5 cm and fully expanded leaves, thus establishing a stable sterile system; The MS solid medium is based on MS medium, containing 30 g / L sucrose, 6.5 g / L agar, pH 5.8; Step 2: Directional selection of explants and browning resistance pretreatment Stem or root segments were selected from sterile seedlings as explants: 1.0-1.5 cm long stem segments were cut, retaining 1-2 plump axillary buds, removing aging tissue and excess leaves at the base, leaving only one small leaf at the top; 1.0-1.5 cm long root segments were cut, removing the aging parts at the base; the stem or root segments were immersed in a pretreatment solution and treated in the dark at 25 ℃ for 1-4 hours; the pretreatment solution was 1 / 2 MS liquid medium containing 0.1-0.3 mmol / L silver nitrate and 50-100 mg / L ascorbic acid. Step 3: Preparation of high-efficiency induction medium Using 1 / 2 MS as the basal medium, add the following components: IBA 0.8-1.2 mg / L, NAA 0.2-0.4 mg / L, 6-BA 0.05-0.1 mg / L, sucrose 25-35 g / L, agar 6-7 g / L, and Pro 50-100 mg / L; adjust the pH to 5.7-5.9 with 1 mol / L NaOH or HCl, and autoclave at 121℃ for 20 min before use. Step 4: Segmented induction culture Pretreated stem or root segments were inoculated into a high-efficiency induction medium, with 3-5 segments per 100 mL Erlenmeyer flask. The following stage of induction was used: ① Dark culture stage: culturing in the dark at 22-24 ℃ for 3-5 days; ② Light culture stage: transferring to an environment with a light intensity of 1500-2000 lx, a light duration of 12 h / d, a temperature of 23-25 ​​℃, and a relative humidity of 60-70%, culturing for 25-35 days to induce adventitious root formation. Step 5: Proliferation Culture and Product Accumulation Cut off the induced adventitious roots, retaining 2-3 cm of root tip, and inoculate them into proliferation medium. Place them in a shaker for cultivation: temperature 23-25 ​​℃, light intensity 1000-1500 lx, photoperiod 12 h / d, rotation speed 100-120 r / min, and culture for 20-30 days. The proliferation medium is 1 / 2 MS liquid medium containing IBA 0.5-0.8 mg / L + sucrose 30-40 g / L + hydrolyzed casein 100-200 mg / L.

2. The efficient method for inducing adventitious roots of Edelweiss according to claim 1, characterized in that, Replace steps 2 and 3 with steps 6 and 7: Step 6: Induction of Edelweiss Callus 1) Explants: Leaves, petioles or stem segments from sterile seedlings are used as explants; 2) Induction medium: Based on MS or B5 medium, add auxin, cytokinin, sucrose 30 g / L, agar 6-8 g / L, and adjust the pH to 5.6-5.8; the auxin is 1.0-2.0 mg / L 2,4-D or 0.5-1.5 mg / L NAA; the cytokinin is 0.1-0.5 mg / L 6-BA; 3) Culture: Explants were inoculated onto induction medium and cultured in the dark at 25°C for 20-30 days until loose, pale yellow or light green granular callus tissue was formed; subcultured twice every 2-3 weeks with subculture medium; the subculture medium was based on MS medium supplemented with 0.5-1.0 mg / L 2,4-D and 0.1-0.5 mg / L 6-BA. Step 7: High-efficiency induction culture Callus tissue was inoculated onto a high-efficiency induction medium and induced for 45 days to obtain adventitious roots induced from the callus tissue, which were then used for segmented induction culture.

3. The application of the method according to claim 1 or 2 in improving the adventitious root induction rate of Edelweiss.

4. The application of the method according to claim 1 or 2 in increasing the content of total flavonoids and leonuronic acid in adventitious roots of Leontopodium distichum.

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