阅读说明：本技术 一种多倍体大花萱草组织培养及培养基 (Polyploid hemerocallis middendorfii tissue culture and culture medium ) 是由 陈曦 刘志洋 汪磊 王金刚 龚束芳 曲彦婷 周晔 鲁耀 刘鑫 李广忻 张立微 于 2021-08-27 设计创作，主要内容包括：本发明提供一种多倍体大花萱草组织培养的方法及培养基,包括以下步骤：(1)外植体的选取与消毒处理；(2)不定芽的诱导培养,将消毒后的外植体接种至诱导培养基上；(3)不定芽的增殖培养,将不定芽接种于不同增殖培养基上；(4)不定芽的生根培养,当不定芽长至2-3cm时,将不定芽分割成单个外植体,以1/2改性MS为基本培养基,添加NAA在生根培养基中；(5)炼苗及移栽；其中,所述培养基为改性MS培养基。本发明筛选出以多倍体大花萱草的分蘖茎段为外植体,直接诱导生成不定芽,缩短了成苗时间,降低了生产成本；加快了多倍体大花萱草繁殖速度,同时能打破季节局限,进而可大批量生产以满足市场需求,实现育苗工厂化生产。(The invention provides a method for tissue culture of polyploid hemerocallis middendorfii and a culture medium, which comprises the following steps: (1) selecting and disinfecting explants; (2) performing induction culture on the adventitious bud, and inoculating the sterilized explant to an induction culture medium; (3) enrichment culture of adventitious buds, namely inoculating the adventitious buds to different enrichment culture media; (4) rooting culture of the adventitious bud, namely when the adventitious bud grows to 2-3cm, dividing the adventitious bud into single explants, taking 1/2 modified MS as a basic culture medium, and adding NAA into the rooting culture medium; (5) hardening and transplanting seedlings; wherein the culture medium is a modified MS culture medium. The method screens out the tillering stem section of the polyploid hemerocallis middendorfii as an explant, directly induces and generates adventitious buds, shortens the seedling time and reduces the production cost; the propagation speed of the polyploid hemerocallis middendorfii is accelerated, the season limitation can be broken, and further mass production can be realized to meet the market demand, and the industrialized production of seedling cultivation is realized.)

1. A method for tissue culture of polyploid hemerocallis middendorfii is characterized by comprising the following steps:

(1) selecting and disinfecting explants: selecting robust tillering stem segments of various polyploid hemerocallis middendorfii as explant materials, cutting the taken tillering stem segments into 1-2cm bud-remaining segments, cleaning the materials with washing powder, washing with running water for 60min, disinfecting with 75% alcohol on a super clean bench for 30s, washing with sterile water for 3 times, sterilizing with 0.1% mercuric chloride for 8min, washing with sterile water for 4 times, and continuously shaking disinfectant in the whole disinfection process;

(2) and (3) induction culture of adventitious buds: inoculating the sterilized explant to an induction culture medium, and performing illumination culture;

(3) and (3) propagation culture of adventitious buds: inoculating the adventitious bud on an enrichment medium, and performing illumination culture;

(4) rooting culture of adventitious buds: when the adventitious bud grows to 2-3cm, the adventitious bud is divided into single explants, the explants are inoculated in a rooting culture medium, the root length is measured after 30 days, and transplanting is carried out;

(5) hardening and transplanting seedlings: opening a tissue culture bottle cap when the root grows to 2-3cm, hardening seedlings at room temperature for 3-4 days, irradiating by sunlight, washing out the residual culture medium at the root, washing the root with 0.1% carbendazim liquid medicine, and directly transplanting to turfy soil: vermiculite = 1: 1, watering the nutrition pot with enough root fixing water, and then watering for 1 time by a spraying method every day, wherein the temperature is controlled to be 20-30 ℃, and the humidity is 80-90 percent to obtain a finished product;

the permeation culture medium and the multiplication culture medium are both a modified MS culture medium +6-BA + NAA;

the rooting culture medium is 1/2 modified MS culture medium + NAA;

the concentration of the modified MS culture medium is mg/L, and the modified MS culture medium comprises the following components:

MS culture medium, modified antibacterial agent, 1000 times diluted triadimefon and 10% thiamethoxam;

the preparation method of the modified antibacterial agent comprises the following steps: adding 0.1-0.5 part of benzimidazole and 0.1-0.5 part of isothiophene into 100mL of absolute ethanol, stirring for dissolving, then adding 5-10 parts of activated carbon, reacting at 60 ℃ for 1-2h, filtering after the reaction is finished, and drying to obtain the modified antibacterial agent.

2. The method of claim 1, wherein the variety of the polyploid hemerocallis middendorfii is one of ruby, lucky, orange, pink, big cup, and dolls.

3. The method of claim 1, wherein the permeable medium in step (2) is modified MS Medium 1L +6-BA1mg/L + NAA0.1mg/L.

4. The method of claim 1, wherein the inducing culture medium of step (2) further comprises adding sucrose 30g/L and agar 7g/L, and adjusting pH to 6-7.

5. The method for tissue culture of polyploid hemerocallis middendorfii as claimed in claim 1, wherein the culture temperature in step (2) is 20-30 ℃, the relative humidity of air is controlled at 40-60%, the illumination intensity is 1000-.

6. The method of claim 1, wherein the multiplication medium in step (3) is modified MS medium 1L +6-BA1-2mg/L + NAA 0.1-0.2mg/L, and further comprises modified chitosan aerogel.

7. The method of claim 6, wherein the modified chitosan aerogel alkalinity is prepared by the steps of: dissolving 20 parts by mass of chitosan in 1000 parts by volume of 2% acetic acid solution, performing rotary evaporation concentration at 75 ℃ to 100 parts by volume, pouring the obtained chitosan acetic acid solution into a container, then adding 10 parts by volume of coconut milk, uniformly stirring, and pre-freezing at-20 ℃ for 24 hours to obtain the modified chitosan aerogel.

8. The method as claimed in claim 1, wherein the illumination intensity in step (3) is 2000-3000lx, the temperature is 25 ℃, and the cultivation time is 15-20 days.

9. The method for tissue culture of polyploid hemerocallis middendorfii as claimed in claim 1, wherein the rooting medium in step (4) is 1/2 modified MS medium 1L + NAA 0.1-0.3 mg/L.

10. The method of claim 1, wherein said modified MS medium is supplemented with 1-5g modified antimicrobial, 5-10g triadimefon diluted 1000 fold and 50-100mg 10% thiamethoxam per liter of MS medium.

Technical Field

The invention belongs to the technical field of flower cultivation, and particularly relates to a polyploid hemerocallis middendorfii tissue culture medium.

Background

With the rapid development of social economy, the requirements of people on the surrounding living environment are improved along with the improvement of the living standard of the material culture of people. Therefore, the ornamental value of landscaping and courtyard landscape gardens is improved, garden flowers are required to be attractive and pleasing, and the garden flowers and plants are required to be simple to cultivate, convenient to manage and strong in resistance. Therefore, in recent years, day lily plants begin to be pretty in the garden market, and due to the fact that flowers are large and beautiful, colors are rich, cultivation is simple, management is convenient, spring germination is early, green leaves grow into clumps, and clumping planting and solitary planting can form a landscape with extremely high ornamental value under the shade of green and on the roadside.

The polyploid hemerocallis middendorfii is extremely cold-resistant, drought-resistant and has low requirement on soil, and can grow and bloom on saline-alkali soil. The flower color is bright, beautiful and long in flowering phase, and the flower is started from 6 middle ten days to winter; the flower leaves grow in a bunch shape, the height of the calyx is 10-15 cm, and flowers are arranged above the flower leaves. The ornamental value is very high no matter the leaves are observed or the flowers are observed. Can be planted in a sheet, a flower stem, a flower bed and a pot. The winter can be safely overwintering no matter how cold, the original American product has short time for being introduced into China. The polyploid hemerocallis middendorfii directionally selects fine varieties with the characteristics of gorgeous flower color, beautiful flower appearance, large flower size, high flower laying rate and the like as parents for hybridization propagation, and the obtained F1 generation seedlings have excellent hybridization advantages of vigorous growth vigor, more gorgeous flower color and the like for the most part. Therefore, each merchant is introduced and bred abroad, but the cost is high, the resources are limited, and the phenomenon of short supply and demand in the market is caused.

At present, hemerocallis middendorfii is valued by gardening in various countries in the world. In developed countries, hemerocallis middendorfii has been widely applied to greening and beautifying public greenbelts, parks, squares and courtyards and preventing water and soil loss in recent ten years, and has achieved higher greening and beautifying effects. Due to the excellent characteristics and wide market development prospect, Chinese investors are attracted to perform germplasm collection, breeding and seedling production, and more than one thousand varieties are put into the market at present. At present, China fully realizes that hemerocallis middendorfii is an excellent ground cover plant, resources of China are rich, and the hemerocallis middendorfii variety breeding and breeding work is carried out, so that the important economic and social benefits are achieved. In 70 s, the plant research of Chinese academy of sciences is carried out in Beijing and Guangzhou to carry out variety breeding test, and finally more than one hundred varieties which can be widely cultivated in gardens of China are bred, and hemerocallis middendorfii is one of the varieties. A large number of experiments prove that the hemerocallis middendorfii is an excellent flower plant material.

At present, the propagation of polyploid hemerocallis mainly takes the division as the main part, only 4-5 hemerocallis can be propagated from 1 hemerocallis every year, large land and cultivation stock plants are needed, the propagation coefficient is low, the period is long, and the application speed of new varieties is limited. The tissue culture method can greatly improve the propagation coefficient, can produce a large amount of seedlings in a short time and put the seedlings on the market, and quickens the application pace of new varieties.

Chinese patent (CN 201310405775: a tissue culture method and culture medium for polyploid hemerocallis middendorfii) can rapidly propagate polyploid hemerocallis middendorfii through tissue culture, the production period only needs about 2 months, but the problems of induction rate, proliferation rate, rooting rate and the like of adventitious buds during tissue culture are not concerned, and only statistics is carried out on the subsequent transplanting rate.

Disclosure of Invention

Aiming at the defects of the prior art, the invention provides a method for tissue culture of polyploid hemerocallis middendorfii.

In order to achieve the purpose, the invention provides the following technical scheme:

a method for tissue culture of polyploid hemerocallis middendorfii is characterized by comprising the following steps:

(1) selecting and disinfecting explants: selecting robust tillering stem segments of various polyploid hemerocallis middendorfii as explant materials, cutting the taken tillering stem segments into 1-2cm bud-remaining segments, cleaning the materials with washing powder, washing with running water for 60min, disinfecting with 75% alcohol on a super clean bench for 30s, washing with sterile water for 3 times, sterilizing with 0.1% mercuric chloride for 8min, washing with sterile water for 4 times, and continuously shaking disinfectant in the whole disinfection process;

(2) and (3) induction culture of adventitious buds: inoculating the sterilized explant to an induction culture medium, and performing illumination culture;

(3) and (3) propagation culture of adventitious buds: inoculating the adventitious bud on an enrichment medium, and performing illumination culture;

(4) rooting culture of adventitious buds: when the adventitious bud grows to 2-3cm, the adventitious bud is divided into single explants, the explants are inoculated in a rooting culture medium, the root length is measured after 30 days, and transplanting is carried out;

(5) hardening and transplanting seedlings: opening a tissue culture bottle cap when the root grows to 2-3cm, hardening seedlings at room temperature for 3-4 days, irradiating by sunlight, washing out the residual culture medium at the root, washing the root with 0.1% carbendazim liquid medicine, and directly transplanting to turfy soil: 1, vermiculite: 1, watering the nutrition pot with enough root fixing water, and then watering for 1 time by a spraying method every day, wherein the temperature is controlled to be 20-30 ℃, and the humidity is 80-90 percent to obtain a finished product;

the permeation culture medium and the multiplication culture medium are both a modified MS culture medium +6-BA + NAA;

the rooting culture medium is 1/2 modified MS culture medium + NAA;

the concentration of the modified MS culture medium is mg/L, and the modified MS culture medium comprises the following components:

MS culture medium, isothiazole, benzimidazole, triadimefon diluted 1000 times and 10% thiamethoxam.

The preparation method of the modified antibacterial agent comprises the following steps: adding 0.1-0.5 part of benzimidazole and 0.1-0.5 part of isothiophene into 100mL of absolute ethanol, stirring for dissolving, then adding 5-10 parts of activated carbon, reacting at 60 ℃ for 1-2h, filtering after the reaction is finished, and drying to obtain the modified antibacterial agent.

Preferably, the variety of the polyploid hemerocallis middendorfii is one of ruby, red fortune, orange yellow, pink flesh, big golden cup and golden doll.

Preferably, the permeable medium in the step (2) is modified MS medium 1L +6-BA1mg/L + NAA0.1mg/L.

Preferably, the induction culture medium in the step (2) further comprises the steps of adding 30g/L of sucrose and 7g/L of agar, and adjusting the pH value to 6-7.

Preferably, the culture temperature in the step (2) is 20-30 ℃, the relative air humidity is controlled to be 40-60%, the illumination intensity is 1000-.

Preferably, the proliferation culture medium in the step (3) is a modified MS culture medium 1L +6-BA1-2mg/L + NAA 0.1-0.2mg/L, and chitosan aerogel is also added.

Preferably, the chitosan aerogel alkalinity is prepared by the following steps: dissolving 20 parts by mass of chitosan in 1000 parts by volume of 2% acetic acid solution, performing rotary evaporation concentration at 75 ℃ to 100 parts by volume, pouring the obtained chitosan acetic acid solution into a container, then adding 10 parts by volume of coconut milk, uniformly stirring, pre-freezing at-20 ℃ for 24 hours, taking out, and performing freeze drying at-55 ℃ for 36 hours to obtain the chitosan aerogel.

Preferably, the illumination intensity in the step (3) is 2000-3000lx, the temperature is 25 ℃, and the culture time is 15-20 days.

Preferably, the rooting culture medium in the step (4) is 1/2 modified MS culture medium 1L + NAA 0.1-0.3 mg/L.

Preferably, 1-5g of modified antibacterial agent, 5-10g of triadimefon diluted 1000 times and 50-100mg of 10% thiamethoxam are added into each liter of MS culture medium in the modified MS culture medium.

Compared with the prior art, the invention has the following beneficial effects:

(1) according to the invention, the MS culture medium is modified, and the modified antibacterial agent is added, wherein the activated carbon in the modified antibacterial agent can provide a dark environment, so that the decomposition of nutrient substances and regulating substances in the culture medium is avoided, the stability and the activity of the culture medium are ensured, and phenol substances in-vitro culture can be adsorbed, so that polyphenol oxidase and peroxidase are inactivated, and the browning of plants is prevented; meanwhile, the benzimidazole and the isothiazole loaded in the active carbon can play a role in sterilization, reduce the pollution rate of the adventitious bud in the processes of induced growth, proliferation growth and rooting growth, and improve the survival rate of the adventitious bud. The triadimefon and the thiamethoxam are added into the culture medium to serve as insecticides, so that the insecticide can enter the hemerocallis middendorfii during the growth of adventitious buds, and the infection and pest damage caused by the hemerocallis middendorfii after the hemerocallis middenfii is transplanted outdoors in the later period can be reduced.

(2) NAA is added in three culture stages, wherein the NAA is a high-efficiency plant growth regulator and has small toxic and side effects, and the NAA can improve the contents of chlorophyll, protein and nucleic acid in a plant and photosynthetic efficiency, improve the activities of peroxidase and nitrate reductase, promote the carbon and nitrogen metabolism of the plant and promote the division and the elongation of plant cells, so that the absorption of the plant to nutrients and the accumulation of dry substances are enhanced, and the growth of the hemerocallis middendorfii is promoted. The culture medium has different formulas in different stages, is the optimal combination for culturing the polyploid hemerocallis middendorfii for large-scale propagation, can rapidly propagate a large number of tissue culture seedlings, and meets the requirements of landscaping markets.

(3) According to the invention, the modified chitosan aerogel is added into the culture medium in the enrichment culture, wherein the chitosan is used as an organic antibacterial agent, and the pollution rate of the culture medium is further reduced due to the antibacterial property of the chitosan; meanwhile, the added coconut milk is used as an organic additive, can provide necessary physiological active substances and trace components for plant growth, and can play a role in remarkably improving differentiation rate, regenerating seedling height and strengthening seedlings in the proliferation stage of plant tissue culture as an additive for perfecting a plant tissue culture regeneration system.

(4) The method screens out the tillering stem section of the polyploid hemerocallis middendorfii as an explant, can directly induce and generate adventitious buds, accelerates the propagation speed of the polyploid hemerocallis middendorfii, can break season limitation, further can realize mass production to meet market demands, and realizes seedling culture industrialized production.

Detailed Description

The technical solutions of the present invention will be described clearly and completely with reference to specific embodiments, and it should be understood 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 method for tissue culture of polyploid hemerocallis middendorfii is characterized by comprising the following steps:

(1) preparation of modified MS culture medium: adding 0.1g of benzimidazole and 0.1g of isothiophene into 100mL of absolute ethanol, stirring for dissolving, then adding 5g of activated carbon, reacting at 60 ℃ for 1h, filtering after the reaction is finished, and drying to obtain a modified antibacterial agent; and then 1L of MS culture medium is taken, 1g of modified antibacterial agent, 5g of 20% triadimefon missible oil diluted by 1000 times and 50mg of 10% thiamethoxam are added, and the modified MS culture medium is obtained after uniform mixing.

(2) Selecting and disinfecting explants: selecting a robust tillering stem section of the red gem polyploid hemerocallis middendorfii as an explant material, cutting the taken tillering stem section into 1cm bud-remaining small sections, cleaning the material with washing powder, then washing with running water for 60min, disinfecting with 75% alcohol for 30s on an ultraclean workbench, washing with sterile water for 3 times, then sterilizing with 0.1% mercuric chloride for 8min, washing with the sterile water for 4 times, and continuously shaking disinfectant in the whole disinfection process.

(3) And (3) induction culture of adventitious buds: inoculating the sterilized explants to an induction culture medium, inoculating 10 bottles of explants to each bottle of 3 explants, wherein the formula of the induction culture medium is a modified MS culture medium 1L +6-BA1mg/L + NAA0.1mg/L, simultaneously adding 30g/L of sucrose and 7g/L of agar, adjusting the pH value to 6, controlling the culture temperature to be 20 ℃, controlling the relative air humidity to be 40%, and controlling the illumination intensity to be 1000lx, and culturing for 15 days.

(4) And (3) propagation culture of adventitious buds: the modified chitosan aerogel is put into a multiplication culture medium, then the adventitious buds are inoculated on the multiplication culture medium, 3 buds are inoculated in each bottle, 10 bottles are inoculated, the formula of the multiplication culture medium is a modified MS culture medium 1L +6-BA1mg/L + NAA0.1mg/L, the illumination culture is carried out for 20 days, the illumination intensity is 2000lx, and the temperature is 25 ℃.

(5) Rooting culture of adventitious buds: when the adventitious bud grows to 2cm, the adventitious bud is divided into single explants, the explants are inoculated to a rooting medium, 3 seedlings are cultured in each bottle, 10 bottles are inoculated, the formula of the rooting medium is 1/2 modified MS medium 1L + NAA0.1mg/L, the root length is measured after 30 days, and transplanting is carried out.

(6) Hardening and transplanting seedlings: opening a tissue culture bottle cap when the root grows to 2cm, hardening seedlings at room temperature for 3 days, irradiating by sunlight, washing out the residual culture medium at the root, washing the root with 0.1% carbendazim liquid medicine, and directly transplanting to turfy soil: 1, vermiculite: 1, watering the soil in the nutrition pot sufficiently, and then watering for 1 time by a spraying method every day, wherein the temperature is controlled at 20 ℃, and the humidity is 80 percent to obtain a finished product.

Example 2

A method for tissue culture of polyploid hemerocallis middendorfii is characterized by comprising the following steps:

(1) preparation of modified MS culture medium: adding 0.2g of benzimidazole and 0.2g of isothiophene into 100mL of absolute ethanol, stirring for dissolving, then adding 8g of activated carbon, reacting at 60 ℃ for 1.5h, filtering after the reaction is finished, and drying to obtain a modified antibacterial agent; and then taking 1LMS culture medium, adding 2g of modified antibacterial agent, 7g of 20% triadimefon missible oil diluted by 1000 times and 60mg of 10% thiamethoxam, and uniformly mixing to obtain the modified MS culture medium.

(2) Selecting and disinfecting explants: selecting a robust tillering stem section of red transport polyploid hemerocallis middendorfii as an explant material, cutting the adopted tillering stem section into 1cm bud-remaining small sections, cleaning the material with washing powder, then washing with running water for 60min, disinfecting with 75% alcohol for 30s on an ultraclean workbench, washing with sterile water for 3 times, then sterilizing with 0.1% mercuric chloride for 8min, washing with sterile water for 4 times, and continuously shaking disinfectant in the whole disinfection process.

(3) And (3) induction culture of adventitious buds: inoculating the sterilized explants to an induction culture medium, inoculating 10 bottles of explants to 3 per bottle, wherein the formula of the induction culture medium is a modified MS culture medium 1L +6-BA1mg/L + NAA0.1mg/L, simultaneously adding 30g/L of sucrose and 7g/L of agar, adjusting the pH value to 6.5, controlling the culture temperature to be 25 ℃, controlling the relative air humidity to be 50%, controlling the illumination intensity to be 1500lx, and culturing for 15 days.

(4) And (3) propagation culture of adventitious buds: putting the modified chitosan aerogel into a multiplication culture medium, then inoculating the adventitious buds on different multiplication culture media, wherein each bottle has 3 buds, inoculating 10 bottles, the formula of the multiplication culture medium is modified MS culture medium 1L +6-BA 1.2mg/L + NAA0.12mg/L, and the multiplication culture medium is cultured for 20 days under illumination with the illumination intensity of 2500lx and the temperature of 25 ℃.

(5) Rooting culture of adventitious buds: when the adventitious bud grows to 2.5cm, the adventitious bud is divided into single explants, the explants are inoculated to a rooting medium, 3 seedlings are cultured in each bottle, 10 bottles are inoculated, the formula of the rooting medium is 1/2 modified MS medium 1L + NAA0.15mg/L, the root length is measured after 30 days, and the explants are transplanted.

(6) Hardening and transplanting seedlings: opening a tissue culture bottle cap when the root grows to 2.5cm, hardening seedlings at room temperature for 3 days, irradiating by sunlight, washing out the residual culture medium at the root, washing the root with 0.1% carbendazim liquid medicine, and directly transplanting to turfy soil: 1, vermiculite: 1, watering the nutrient pot with enough root fixing water, and then watering for 1 time by a spraying method every day, wherein the temperature is controlled at 25 ℃, and the humidity is 85 percent, so as to obtain a finished product.

Example 3

A method for tissue culture of polyploid hemerocallis middendorfii is characterized by comprising the following steps:

(1) preparation of modified MS culture medium: adding 0.4g of benzimidazole and 0.4g of isothiophene into 100mL of absolute ethanol, stirring for dissolving, then adding 9g of activated carbon, reacting for 2 hours at 60 ℃, filtering after the reaction is finished, and drying to obtain a modified antibacterial agent; and then taking 1L of MS culture medium, adding 4g of antibacterial agent, 9g of 20% triadimefon missible oil diluted by 1000 times and 80mg of 10% thiamethoxam, and uniformly mixing to obtain the modified MS culture medium.

(2) Selecting and disinfecting explants: selecting a strong tillering stem section of orange polyploid hemerocallis middendorfii as an explant material, cutting the adopted tillering stem section into bud-remaining small sections of 2cm, cleaning the material with washing powder, then washing with running water for 60min, disinfecting with 75% alcohol for 30s on an ultraclean workbench, washing with sterile water for 3 times, then sterilizing with 0.1% mercuric chloride for 8min, washing with sterile water for 4 times, and continuously shaking disinfectant in the whole disinfection process.

(3) And (3) induction culture of adventitious buds: inoculating the sterilized explants to an induction culture medium, inoculating 10 bottles of explants to each bottle of 3 explants, wherein the formula of the induction culture medium is a modified MS culture medium 1L +6-BA1mg/L + NAA0.1mg/L, simultaneously adding 30g/L of sucrose and 7g/L of agar, adjusting the pH value to 6.5, controlling the culture temperature to be 28 ℃, controlling the relative air humidity to be 55%, controlling the illumination intensity to be 1500lx, and culturing for 15 days.

(4) And (3) propagation culture of adventitious buds: the modified chitosan aerogel is put into a multiplication culture medium, then adventitious buds are inoculated on different multiplication culture media, 3 buds are inoculated in each bottle, 10 bottles are inoculated, the formula of the multiplication culture medium is modified MS culture medium 1L +6-BA 1.5mg/L + NAA0.15mg/L, the illumination culture is carried out for 20 days, the illumination intensity is 2500lx, and the temperature is 25 ℃.

(5) Rooting culture of adventitious buds: when the adventitious bud grows to 2.5cm, the adventitious bud is divided into single explants, the explants are inoculated to a rooting medium, 3 seedlings are cultured in each bottle, 10 bottles are inoculated, the formula of the rooting medium is 1/2 modified MS medium 1L + NAA 0.25mg/L, the root length is measured after 30 days, and the seedlings are transplanted.

(6) Hardening and transplanting seedlings: opening a tissue culture bottle cap when the root grows to 2.5cm, hardening seedlings at room temperature for 4 days, irradiating by sunlight, washing out the residual culture medium at the root, washing the root with 0.1% carbendazim liquid medicine, and directly transplanting to turfy soil: 1, vermiculite: 1, watering the nutrient pot with enough root fixing water, and then watering for 1 time by a spraying method every day, wherein the temperature is controlled at 25 ℃, and the humidity is 85 percent, so as to obtain a finished product.

Example 4

A method for tissue culture of polyploid hemerocallis middendorfii is characterized by comprising the following steps:

(1) preparation of modified MS culture medium: adding 0.5g of benzimidazole and 0.5g of isothiophene into 100mL of absolute ethanol, stirring for dissolving, then adding 10g of activated carbon, reacting for 2 hours at 60 ℃, filtering after the reaction is finished, and drying to obtain a modified antibacterial agent; and then taking 1L of MS culture medium, adding 5g of modified antibacterial agent, 10g of 20% triadimefon missible oil diluted by 1000 times and 100mg of 10% thiamethoxam, and uniformly mixing to obtain the modified MS culture medium.

(2) Selecting and disinfecting explants: selecting a robust tillering stem section of the polyploid hemerocallis middendorfii as an explant material, cutting the taken tillering stem section into bud-remaining small sections of 2cm, cleaning the material with washing powder, then washing with running water for 60min, disinfecting with 75% alcohol for 30s on an ultraclean workbench, washing with sterile water for 3 times, then sterilizing with 0.1% mercuric chloride for 8min, washing with the sterile water for 4 times, and continuously shaking disinfectant in the whole disinfection process.

(3) And (3) induction culture of adventitious buds: the modified chitosan aerogel is put into a proliferation culture medium, then the disinfected explants are inoculated onto an induction culture medium, 3 explants are inoculated into 10 bottles in each bottle, the formula of the induction culture medium is 1L +6-BA1mg/L + NAA0.1mg/L of the modified MS culture medium, simultaneously 30g/L of sucrose and 7g/L of agar are added, the pH value is adjusted to 7, the culture temperature is 30 ℃, the relative humidity of air is controlled at 60%, the illumination intensity is 2000lx, and the culture is carried out for 15 days.

(4) And (3) propagation culture of adventitious buds: inoculating the adventitious buds to different proliferation culture media, wherein each bottle contains 3 buds and 10 bottles of the adventitious buds, the formula of the proliferation culture media is modified MS culture medium 1L +6-BA2mg/L + NAA0.2mg/L, and the proliferation culture media are cultured for 20 days under illumination, the illumination intensity is 3000lx, and the temperature is 25 ℃.

(5) Rooting culture of adventitious buds: when the adventitious bud grows to 3cm, the adventitious bud is divided into single explants, the explants are inoculated to a rooting medium, 3 seedlings are cultured in each bottle, 10 bottles are inoculated, the formula of the rooting medium is 1/2 modified MS medium 1L + NAA 0.3mg/L, the root length is measured after 30 days, and transplanting is carried out.

(6) Hardening and transplanting seedlings: opening a tissue culture bottle cap when the root grows to 3cm, hardening seedlings at room temperature for 4 days, irradiating by sunlight, washing out the residual culture medium at the root, washing the root with 0.1% carbendazim liquid medicine, and directly transplanting to turfy soil: 1, vermiculite: 1, watering the nutrient pot with enough root fixing water, and then watering for 1 time by a spraying method every day, wherein the temperature is controlled at 25 ℃, and the humidity is 85 percent, so as to obtain a finished product.

Comparative example 1

A method for tissue culture of polyploid hemerocallis middendorfii is characterized by comprising the following steps:

(1) selecting and disinfecting explants: selecting a robust tillering stem section of the red gem polyploid hemerocallis middendorfii as an explant material, cutting the taken tillering stem section into 1cm bud-remaining small sections, cleaning the material with washing powder, then washing with running water for 60min, disinfecting with 75% alcohol for 30s on an ultraclean workbench, washing with sterile water for 3 times, then sterilizing with 0.1% mercuric chloride for 8min, washing with the sterile water for 4 times, and continuously shaking disinfectant in the whole disinfection process.

(2) And (3) induction culture of adventitious buds: inoculating the sterilized explants to an induction culture medium, inoculating 10 bottles of explants to each bottle of 3 explants, wherein the formula of the culture medium is 1L +6-BA1mg/L + NAA0.1mg/L of MS culture medium, simultaneously adding 30g/L of sucrose and 7g/L of agar, adjusting the pH value to 6, controlling the culture temperature to be 20 ℃, controlling the relative air humidity to be 40%, and controlling the illumination intensity to be 1000lx, and culturing for 15 days.

(3) And (3) propagation culture of adventitious buds: inoculating adventitious buds to different proliferation culture media, wherein each bottle contains 3 buds and 10 bottles, the formula of the culture media is MS culture medium 1L +6-BA1mg/L + NAA0.1mg/L, the illumination culture is carried out for 20 days, the illumination intensity is 2000lx, and the temperature is 25 ℃.

(4) Rooting culture of adventitious buds: when the adventitious bud grows to 2cm, the adventitious bud is divided into single explants, the explants are inoculated to a rooting medium, 3 seedlings are cultured in each bottle, 10 bottles are inoculated, the formula of the rooting medium is 1/2MS medium 1L + NAA0.1mg/L, the root length is measured after 30 days, and the explants are transplanted.

(5) Hardening and transplanting seedlings: opening a tissue culture bottle cap when the root grows to 2cm, hardening seedlings at room temperature for 3 days, irradiating by sunlight, washing out the residual culture medium at the root, washing the root with 0.1% carbendazim liquid medicine, and directly transplanting to turfy soil: 1, vermiculite: 1, watering the soil in the nutrition pot sufficiently, and then watering for 1 time by a spraying method every day, wherein the temperature is controlled at 20 ℃, and the humidity is 80 percent to obtain a finished product.

Comparative example 2

A method for tissue culture of polyploid hemerocallis middendorfii is characterized by comprising the following steps:

(1) preparation of modified MS culture medium: adding 0.1g of benzimidazole and 0.1g of isothiophene into 100mL of absolute ethanol, stirring for dissolving, then adding 5g of activated carbon, reacting at 60 ℃ for 1h, filtering after the reaction is finished, and drying to obtain a modified antibacterial agent; and then 1LMS culture medium is added with 1g of modified antibacterial agent, 5g of 20% triadimefon missible oil diluted by 1000 times and 50mg of 10% thiamethoxam, and the mixture is uniformly mixed to obtain the modified MS culture medium.

(2) Selecting and disinfecting explants: selecting a robust bud of the red jewel polyploid hemerocallis middendorfii as an explant material, cleaning the material with washing powder, then flushing with running water for 60min, disinfecting with 75% alcohol on a superclean bench for 30s, flushing with sterile water for 3 times, then sterilizing with 0.1% mercury bichloride for 8min, flushing with sterile water for 4 times, and continuously shaking the disinfectant in the whole disinfection process.

(3) And (3) induction culture of adventitious buds: inoculating the sterilized explants to an induction culture medium, inoculating 10 bottles of 3 explants per bottle, wherein the formula of the induction culture medium is a modified MS culture medium 1L +6-BA1mg/L + NAA0.1mg/L, adding 30g/L of sucrose and 7g/L of agar, adjusting the pH value to 6, controlling the culture temperature to be 20 ℃, controlling the relative air humidity to be 40%, controlling the illumination intensity to be 1000lx, and culturing for 15 days.

(4) And (3) propagation culture of adventitious buds: the modified chitosan aerogel is put into a multiplication culture medium, then the adventitious buds are inoculated on different multiplication culture media, 3 buds are inoculated in each bottle, 10 bottles are inoculated, the formula of the multiplication culture medium is modified MS culture medium 1L +6-BA1mg/L + NAA0.1mg/L, the illumination culture is carried out for 20 days, the illumination intensity is 2000lx, and the temperature is 25 ℃.

(5) Rooting culture of adventitious buds: when the adventitious bud grows to 2cm, the adventitious bud is divided into single explants, the explants are inoculated to a rooting medium, 3 seedlings are cultured in each bottle, 10 bottles are inoculated, the formula of the rooting medium is 1/2 modified MS medium 1L + NAA0.1mg/L, the root length is measured after 30 days, and transplanting is carried out.

(6) Hardening and transplanting seedlings: opening a tissue culture bottle cap when the root grows to 2cm, hardening seedlings at room temperature for 3 days, irradiating by sunlight, washing out the residual culture medium at the root, washing the root with 0.1% carbendazim liquid medicine, and directly transplanting to turfy soil: 1, vermiculite: 1, watering the soil in the nutrition pot sufficiently, and then watering for 1 time by a spraying method every day, wherein the temperature is controlled at 20 ℃, and the humidity is 80 percent to obtain a finished product.

The conditions of the above examples and comparative examples in the culture process are counted, and the conditions of adventitious bud induction, proliferation coefficient and rooting rate are calculated according to the following formulas:

induction rate (%). The number of adventitious buds successfully induced/total number of inoculated medium. times.100%

Multiplication coefficient ═ number of multiplied seedlings/number of inoculated seedlings

The rooting rate is equal to the number of rooted seedlings/the number of inoculated small seedlings multiplied by 100 percent

The specific data are shown in the following table 1:

TABLE 1 Induction Rate, multiplication factor and rooting Rate for tissue culture of polyploid Hemerocallis fulva

Item	Rate of induction	Coefficient of proliferation	Rooting rate
				Example 1	80％	3.8	94％
Example 2	76％	4.2	95％
				Example 3	90％	4.6	98％
Example 4	78％	4.3	94％
				Comparative example 1	67％	1.4	87％
Comparative example 2	70％	2.1	88％

As can be seen from Table 1, the induction rate, the multiplication coefficient and the rooting rate of examples 1-4 are all superior to those of comparative example 1 and comparative example 2, the culture medium used in comparative example 1 is a common MS culture medium, which shows that the culture medium of the invention has different formulas in different stages, is the optimal combination for culturing the large-scale propagation of the polyploid hemerocallis fulva, and can rapidly propagate a large number of tissue culture seedlings; meanwhile, the addition of the antibacterial agent and the pesticide in the culture medium reduces the pollution rate of the culture medium in the tissue culture process, thereby promoting the increase of the induction rate and the multiplication coefficient; the explant selected in the comparative example 2 is a flower bud, and as can be seen from the comparative example 2, the method screens out the tillering stem section of the polyploid hemerocallis fulva to be the explant, can directly induce and generate adventitious buds, accelerates the propagation speed and the propagation rate of the polyploid hemerocallis fulva, and is the best explant selection in the invention.

Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that changes, modifications, substitutions and alterations can be made in these embodiments without departing from the principles and spirit of the invention, the scope of which is defined in the appended claims and their equivalents.