Konjac seed culture method for increasing the content of glucomannan

Abstract

The application discloses a method for improving the content of konjak seed culture of konjak, and comprises the following specific steps: S1, regeneration of konjak seedling; S2, induction of konjak rhizome; S3, regeneration of konjak microsclerotium; S4, proliferation culture of konjak microsclerotium; and S5, seedling emergence and transplanting. The application combines plant tissue culture technology with konjak cell suspension culture system, utilizes konjak bulb to induce rhizome, induces konjak microsclerotium through parenchyma cells of the rhizome cortex, and obtains a large amount of konjak microsclerotium seed with high content of konjak by a proliferation culture medium. The obtained konjak microsclerotium has vigorous vitality, strong germination capacity, is convenient to store, has no dormancy phenomenon, can be sowed at any time, and has wide application prospect.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of rapid propagation of officinal plants, and more particularly to a konjac seed culture method for increasing the content of glucomannan. BACKGROUND

[0002] Konjac is a perennial herb of Amorphophallus Blume in Araceae, which can be used as officinal plants. Its underground corm is its economic organ, and konjac glucomannan (KGM) is its physiological active main body. Konjac is the only major economic crop that can provide a large amount of glucomannan in nature, and has a large market demand. In recent years, the health function of konjac has been recognized, and it has been listed as one of the top ten health foods by WHO. Research has found that konjac has physiological effects such as weight loss, blood sugar reduction, blood lipid reduction, inflammation resistance, improvement of colon mucosa function, and improvement of immunity.

[0003] In production, corms and rhizomes are often used for asexual propagation. Due to the problems of degeneration of konjac seed balls, low propagation coefficient, and damage by pests and diseases, the yield of konjac has been decreasing year by year, which cannot meet the market demand. Therefore, plant tissue culture technology can be used to produce high-quality konjac seedlings with the help of konjac microcorms, and a faster and more convenient propagation method in tissue culture can be found.

[0004] Konjac microcorms are a kind of micro metamorphic propagation organs, which can obtain seed konjac through embryonic cell proliferation and achieve complete detoxification effect, thereby reducing the problems of degeneration of germplasm, yield reduction, and quality decline caused by vegetative propagation. Konjac microcorms have the advantages of small size, easy storage and transportation, easy seedling growth, and easy rooting, and are expected to replace test tube seedlings in konjac seed propagation.

[0005] Glucomannan, as the main storage material in konjac corms, is a high molecular heteropolysaccharide formed by connecting D-glucose and D-mannose in a certain molecular ratio through β-1,4 glycosidic bond, accounting for more than 50% of the dry basis of konjac corms. The content of glucomannan is a key indicator for measuring the quality of konjac corms. Therefore, increasing the content of glucomannan in konjac microcorms in the rapid propagation system is crucial for preparing konjac seeds with high propagation potential. SUMMARY

[0006] Therefore, the present application aims to provide a konjac regeneration microcorm rapid propagation system, which induces microcorms from rhizomes and obtains a large number of konjac microcorm seeds with high glucomannan content through corm proliferation, thereby realizing the preparation of excellent original seeds.

[0007] In order to achieve the above-mentioned purpose, the present application realizes the following technical scheme:

[0008] The application discloses a konjak seed culture method for increasing the content of konjak.

[0009] S1. Konjak seedling regeneration: konjak tubers are inoculated in bud culture medium to induce adventitious buds; the bud culture medium is as follows: 1 / 2-1 MS, 0.4-0.6% konjak powder, 2-3% sucrose, 2.0 mg / L 6-BA, 0.2 mg / L NAA, 0.2-0.5 mg / L TDZ, 0.1-0.2% agar powder, 0.05% activated carbon, and pH 5.8-6.0;

[0010] S2. Konjak rhizome induction: the adventitious buds and the base of the adventitious buds induced in S1 are separated from the bulbs, and then the separated adventitious buds and the base thereof are transferred into rhizome induction medium to induce the base of the adventitious buds to swell and form thick rhizomes; the rhizome induction medium is as follows: modified MS, 0.5-1.0% mannose, 1-3% sucrose, 0.1-0.3% mannitol, 1.0-2.0 mg / L 6-BA, 0.2-1.0 mg / L NAA, 0.1-0.3 mg / L KT, 0.4-0.6% agar powder, and pH 5.8-6.0; the modified MS is obtained by adjusting the molar ratio of NO3 - to NH4 + to (3-5):1 in the MS medium, and the total nitrogen source concentration is unchanged;

[0011] S3. Konjak microbulb induction: the thick rhizomes and the base of the adventitious buds induced in S2 are separated along the swelling part, and 1 / 2 of the aerial roots differentiated near the bud points are removed, then the rhizomes and the bulbs are cut into small pieces and transferred into microbulb induction medium to perform liquid suspension culture, so as to induce konjak microbulbs; the microbulb induction medium is as follows: MS, 0.5-1.0% mannose, 2-3% sucrose, 0.1-0.3% mannitol, 2.0 mg / L 6-BA, 0.2 mg / L NAA, 0.1-0.5 mg / L ZT, and 1.0 mg / L arginine, and pH 5.8-6.0;

[0012] S4. Konjak microbulb proliferation culture: the konjak microbulbs induced in S3 are cut on the epidermis to form wounds, the bud points are upward, and the konjak microbulbs are half-buried into microbulb proliferation medium to promote the konjak microbulbs to proliferate a large number of konjak microbulbs; the microbulb proliferation medium is as follows: modified B5, 0.5-2.0% mannose, 2-3% sucrose, 0.1-0.2% konjak powder, 0.1-0.3% mannitol, 1.0 mg / L 6-BA, 1.0 mg / L NAA, 0.1-0.5 mg / L TDZ, 0.3-0.5% agar powder, 1.0 mg / L glycine and 1.0 mg / L arginine, and pH 5.8-6.0; the modified B5 is obtained by adding 0.33 g / L NH4NO3 to the B5 medium.

[0013] S5. Seedling transplanting: the microsclerotia of the konjac proliferated in S4 are soaked in 25 mg / L SA for 1 h, then cut into multiple microsclerotia along the connecting part, the size and morphology of the microsclerotia are counted, and the microsclerotia seeds are sowed in a substrate with a volume ratio of 6:3:1 of nutrient soil:vermiculite:perlite, and the germination rate is counted after 1 month.

[0014] Preferably, the culture conditions of step S1 include a light intensity of 1500 lx, a light time of 12 h / d, a light temperature of 23-25℃, a dark temperature of 20-22℃, a culture time of 25-30 d, and a pH of 5.9.

[0015] Preferably, the culture conditions of step S2 include a light intensity of 1000 lx, a light time of 8 h / d, a light temperature of 23-25℃, a dark temperature of 20-22℃, a culture time of 25-30 d, and a pH of 5.9.

[0016] Preferably, step S3 is carried out in a dark environment.

[0017] More preferably, the culture conditions of step S3 include a shaking speed of 100 rpm, a dark culture temperature of 20-22℃, a culture time of 15-20 d, and a pH of 5.8.

[0018] Preferably, the culture conditions of step S4 include a light intensity of 1000 lx, a light time of 12 h / d, a light quality condition of 80-85% red light+15-20% blue light, a light temperature of 25℃, a dark temperature of 22℃, a culture time of 25-30 d, and a pH of 5.9.

[0019] Preferably, in step S2, the sclerotia are cut and half-buried in the rhizome induction medium with the bud point placed obliquely upward.

[0020] Preferably, in step S4, when the size of the induced konjac microsclerotia exceeds 0.6 cm, the new konjac microsclerotia are cut from the original konjac microsclerotia along the connecting part, and the new konjac microsclerotia are inoculated in a microsclerotia proliferation medium with a thickness of 1.5-2 cm for further proliferation culture.

[0021] Preferably, in step S5, the seedling transplanting, the residual medium on the surface of the sclerotia is washed, the sclerotia are then pried into single sclerotia along the connecting part, and the sclerotia are sowed after being air-dried for 6 h in a ventilated place.

[0022] Preferably, the temperature for the seedling transplanting culture is 20-25℃, and the light condition is natural light.

[0023] Further, the application provides a konjac seed culture method for increasing the content of glucomannan, which comprises the following steps:

[0024] S1. Amorphophallus regeneration: Amorphophallus tuber is inoculated in the bud culture medium, and the bud is induced by light culture; the bud culture medium formula is: 0.4%-0.6% amorphophallus powder, 2-3% sucrose, 2 mg / L 6-BA, 0.2 mg / L NAA, 0.2 mg / L TDZ, 0.1%-0.2% agar powder, 1-2 g mannitol, 0.05% activated carbon are added in 1 / 2-1 MS medium, and the pH is adjusted to 5.8-6.0 to induce adventitious buds.

[0025] S2. Amorphophallus rhizome induction: the adventitious buds induced in S1 and the base thereof are separated from the bulb, and are transferred to the rhizome induction medium to induce the adventitious buds to germinate and form thick rhizomes; the rhizome induction medium formula is: 0.5%-1% mannose, 1-3% sucrose, 0.1-0.3% mannitol, 1.0-2.0 mg / L 6-BA, 0.2-1.0 mg / L NAA, 0.1-0.3 mg / L KT, 0.4-0.6% agar powder are added in modified MS (NH4NO3 molar ratio is 5:1), and the pH is 5.8-6.0. 3- / NH4 +

[0026] Preferably, the bulb selected in step S2 is a metamorphic bulb formed by stem swelling at the base of the amorphophallus regeneration seedling.

[0027] S3. Amorphophallus microbulb regeneration: the newly formed rhizome and the original bulb are separated along the swelling part, and 1 / 2 of the aerial roots differentiated from the microbulb are removed, and the rhizome and the bulb are cut into small pieces and transferred to the microbulb induction medium for liquid suspension culture in a dark environment, and the amorphophallus microbulb seeds are obtained; the microbulb induction medium formula is: MS, 0.5%-1% mannose, 2-3% sucrose, 0.1-0.3% mannitol, 2.0 mg / L 6-BA, 0.2 mg / L NAA, 0.1-0.5 mg / L ZT, 0.3%-0.5% agar powder, 1.0 mg / L arginine, and the pH is 5.8-6.0.

[0028] S4. Microbulb proliferation culture: the amorphophallus microbulb obtained in step S3 is cut on the epidermis to form a wound, and is semi-buried in the microbulb proliferation culture medium for proliferation culture under red and blue light to obtain a large amount of amorphophallus microbulb seeds; the microbulb proliferation culture medium formula is: modified B5 (0.33 g / L NH4NO3 is added), 0.5%-2.0% mannose, 2-3% sucrose, 0.1-0.3% mannitol, 1.0 mg / L 6-BA, 1.0 mg / L NAA, 0.1-0.5 mg / L TDZ, 0.4%-0.6% agar powder, 1.0 mg / L glycine, 1.0 mg / L arginine, and the pH is 5.8-6.0.

[0029] ​Preferably, in step S4, when the diameter of the newly formed corms exceeds 0.6 cm, the new corms are separated from the corm body along the corm connection, with the bud point upward, and inoculated into the proliferation medium alone. At the same time, the medium should be poured to a thickness of 1.5-2 cm to facilitate the formation of a large number of microcorms.

[0030] S5. Seedling transplanting: The microcorms of the amorphophallus paeoniifolius in the proliferation culture are soaked in 25 mg / L SA for 1 h, then separated into multiple microcorms along the corm connection, and the corm size and shape are counted, and the corm seeds are sown in a substrate with a volume ratio of 6:3:1 of nutrient soil: vermiculite: perlite, and the germination rate is counted after 1 month.

[0031] Preferably, in the seedling transplanting of step S5, the surface of the corms is washed to remove residual culture medium, then separated into single corms along the corm connection, and sown after air-drying for 6 h in a well-ventilated place.

[0032] Compared with the prior art, the present application has the following beneficial effects:

[0033] 1. The present application utilizes the biological characteristics of amorphophallus paeoniifolius corms, uses cell engineering technology to prepare the rhizomes of amorphophallus paeoniifolius, and produces amorphophallus paeoniifolius corm seeds through a suspension culture system. Compared with the traditional technology of forming microcorms by stem swelling of amorphophallus paeoniifolius seedlings, the new technology has an increase of nearly 100 times in propagation efficiency, significantly reduces production costs, shortens the growth cycle of corm seeds, and solves the problem of original seeds in amorphophallus paeoniifolius production.

[0034] 2. The seeds have high vigor and strong stress resistance: the accumulation of glucomannan in the microcorms is promoted through the proliferation medium, and mature corm seeds are obtained. The results of the seedling transplanting experiment show that the germination rate of the amorphophallus paeoniifolius microcorms prepared by the present application can reach 100%, and the stress resistance is strong, which can be used as the original seed of amorphophallus paeoniifolius planting industry. Subsequently, the amorphophallus paeoniifolius microcorms can be mass-produced using a bioreactor, thereby realizing the preparation of a large number of detoxified amorphophallus paeoniifolius seeds, and fully utilizing the advantages of microcorms such as easy storage and transportation, easy seedling growth, small size, and no dormancy period.

[0035] 3. The means and process of the present application are simple, and related microcorm rapid propagation research requires nearly 4 months for explant- adventitious bud- callus- callus proliferation- induction of microcorms, and is limited by experimental conditions, with a callus browning rate of more than 30%. In the present application, the cycle is about 2 months for explant- rhizome- corm regeneration- corm proliferation, and when amorphophallus paeoniifolius microcorms are obtained, the operation of corm regeneration- corm proliferation can be repeated, achieving a cultivation cycle of every 30 d. In addition, the prepared amorphophallus paeoniifolius microcorms are easy to harvest, and only need to be gently separated from the corm body, washed and drained before sowing, which has a small wound compared with cutting propagation, and is not easy to be infected with amorphophallus paeoniifolius soft rot. BRIEF DESCRIPTION OF DRAWINGS

[0036] In order to more clearly illustrate the technical solutions of the embodiments of the present application, the drawings required by the embodiments will be briefly introduced as follows. Obviously, the drawings in the following description only represent some embodiments of the present application, and all other drawings obtained by those of ordinary skill in the art without creative effort based on the drawings provided should be within the protection scope of the present application.

[0037] Figure 1 is the effect picture of konjac at each stage of the method of the present application.

[0038] Figure 2 is the microsclerotium of konjac at each period before harvest and after sowing of the present application.

[0039] Figure 3 is the safranin-fast green staining of the sclerotium slice and rhizome slice of the present application. DETAILED DESCRIPTION

[0040] The technical solutions in the embodiments of the present application will be described clearly and completely as follows. Obviously, the described embodiments only represent some of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those of ordinary skill in the art without creative effort should be within the protection scope of the present application.

[0041] A konjac seed culture method for increasing the content of glucomannan, comprising the following steps:

[0042] S1. Regeneration of konjac seedling: the flower konjac tuber is inoculated in 1 / 2-1 MS medium, 0.4%-0.6% konjac powder, 2-3% sucrose, 2.0 mg / L 6-BA, 0.2 mg / L NAA, 0.2 mg / L TDZ, 0.1%-0.2% agar powder, 0.05% activated carbon are added, and pH is 5.8-6.0, so as to induce adventitious buds;

[0043] S2. Induction of konjac rhizome: the adventitious buds induced in S1 and the base thereof are separated from the sclerotium, and the separated adventitious buds and adventitious bud base are transferred to the rhizome induction medium, so as to induce the sclerotium slice to swell and form thick and strong rhizome, and the induction medium of the rhizome is: modified MS (NO 3- / NH4 + , molar ratio is 5:1, total nitrogen source concentration is 60 mmol / L), 0.5-1.0% mannose, 1-3% sucrose, 0.1-0.3% mannitol, 1.0-2.0 mg / L 6-BA, 0.2-1 mg / L NAA, 0.1-0.3 mg / L KT, 0.4-0.6% agar powder, and pH is 5.8-6.0;

[0044] S3. Regeneration of Amorphophallus microspheres: The thick rhizome formed in S2 is cut along the swollen part from the original corm, and the aerial roots differentiated from the microspheres are removed by 1 / 2. The thick rhizome and the original corm are cut into small pieces and transferred to the microspheres induction medium for regeneration of microspheres. The microspheres are obtained by liquid suspension culture under dark culture conditions. The microsphere induction medium comprises: MS, 0.5-1.0% mannose, 2-3% sucrose, 0.1-0.3% mannitol, 2.0 mg / L 6-BA, 0.2 mg / L NAA, 0.1-0.5 mg / L ZT, and 1.0 mg / L arginine, with pH being 5.8-6.0.

[0045] S4. Proliferation culture of Amorphophallus microspheres: The Amorphophallus microspheres harvested in step S3 are cut on the epidermis to form a wound, with the bud point upward and half-buried in the microsphere proliferation medium. The proliferation culture is performed under red and blue light for 25-30 days, so that the corms are rapidly swollen and a large number of Amorphophallus microsphere seeds are obtained. The microsphere proliferation medium comprises: modified B5 (with 0.33 g / L NH4NO3 added), 0.5-2.0% mannose, 2-3% sucrose, 0.1-0.2% Amorphophallus powder, 0.1-0.3% mannitol, 1.0 mg / L 6-BA, 1.0 mg / L NAA, 0.1-0.5 mg / L TDZ, 1.0 mg / L glycine, and 1.0 mg / L arginine, with pH being 5.8-6.0.

[0046] S5. Germination and transplanting: The Amorphophallus microspheres obtained by proliferation culture are soaked in 25 mg / L SA for 1 h, then cut into multiple microspheres along the corm connection, and the corm size and shape are counted. After being dried, the microsphere seeds are sown in a substrate with a volume ratio of 6:3:1 of nutrient soil, vermiculite and perlite, and the germination rate is counted after 1 month.

[0047] In some embodiments of the present application, the culture conditions in step S1 include light intensity of 1500 lx, light time of 12 h / d, wherein the temperature is 23-25°C during light and 20-22°C during darkness, the culture is performed for 25-30 days, and the pH is 5.9.

[0048] In some embodiments of the present application, the culture conditions in step S2 include light intensity of 1000 lx, light time of 8 h / d, wherein the temperature is 23-25°C during light and 20-22°C during darkness, the culture is performed for 25-30 days, and the pH is 5.9.

[0049] In some embodiments of the present application, in step S2, the microspheres are inoculated in the rhizome induction medium, half-buried in the medium, and the bud point is placed obliquely upward.

[0050] In some embodiments of the present application, the culture conditions of step S3 include a shaking speed of 100 rpm, a temperature of 20-22℃ in the dark, a culture time of 15-20 days, and a pH of 5.8.

[0051] In some embodiments of the present application, the culture conditions of step S4 include a light intensity of 1000 lx, a light time of 12 h / d, a light quality condition of red light 80-85%+blue light 15-20%, a temperature of 25℃ under light and 22℃ in the dark, and a pH of 5.9.

[0052] In some embodiments of the present application, when the size of the new bulbs exceeds 0.6 cm in step S4, the new bulbs are separated from the original bulbs along the connection between the bulbs, and inoculated into the proliferation medium, and the medium should be poured to a thickness of 1.5-2 cm to facilitate the formation of a large number of microbulbs.

[0053] In some embodiments of the present application, the medium formula in step S1 is: MS, 0.5% konjac powder, 2.0 mg / L 6-BA, 0.2 mg / L NAA, 0.2 mg / L TDZ, 0.15% agar powder, 0.05% activated carbon, 0.2% mannitol, 2.8% sucrose, and pH 5.9.

[0054] The present application uses konjac microbulbs as explants and realizes in vitro culture by artificial technology. Konjac powder is added to the adventitious bud medium to configure a semi-solid medium by using its gelation and water retention, so as to accelerate the diffusion of polyphenols generated by konjac browning in the medium and reduce the browning rate of the explants. In addition, the main component of konjac powder, glucomannan, can be absorbed and utilized by konjac explants, thereby greatly shortening the formation time of microbulbs.

[0055] In some embodiments of the present application, the rhizome induction medium formula in step S2 is: modified MS (NO3 - / NH4 + ratio of 5:1, total nitrogen source concentration of 60 mmol / L), 1.5 mg / L 6-BA, 1.0 mg / L NAA, 0.2 mg / L KT, 0.5% agar powder, 2% sucrose, 0.5% mannose, 0.2% mannitol, and pH 5.9.

[0056] Konjac microbulbs are selected as the induced explants because the epidermis of the bulb, as a seed organ, is rich in embryonic cells, which can form adventitious buds through proliferation and differentiation, and further induce rhizomes, without the need for a step of callus proliferation culture. In addition, there are abundant parenchyma cells under the cortex of konjac rhizomes, which are ideal materials for bulb rapid propagation system.

[0057] In some embodiments of the present application, the microsclerotum induction medium in step S3 is MS, 1% mannose, 2% sucrose, 0.2-0.3% mannitol, 2.0 mg / L 6-BA, 0.2 mg / L NAA, 0.2 mg / L ZT, 1.0 mg / L arginine, and pH 5.8.

[0058] The main storage material in the konjac cormel is glucomannan. By adding mannose and mannitol, the precursors of glucomannan synthesis, in the microsclerotum induction medium, the proliferation and differentiation of the embryonic cell groups can be effectively promoted, the cormel organs with storage function are formed, the induction rate of the microsclerotum is improved, and the konjac original seed with strong propagation potential is cultivated.

[0059] In some embodiments of the present application, the proliferation medium in step S4 is modified B5 (adding 0.33 g / L NH4NO3), 1% mannose, 3% sucrose, 0.2% konjac powder, 0.2% mannitol, 1.0 mg / L 6-BA, 1.0 mg / L NAA, 0.3 mg / L TDZ, 1.0 mg / L glycine, 1.0 mg / L arginine, and pH 5.9.

[0060] By further culturing in the proliferation medium, the accumulation of glucomannan in the microsclerotum can be promoted, the volume is rapidly expanded, the quality standard of the original seed can be rapidly reached after planting, and the propagation ability of the microsclerotum is further improved.

[0061] In some embodiments of the present application, the specific operation of the seedling transplanting in step S5 is that the brown epidermis on the surface of the microsclerotum is gently scraped off with scissors, the residual medium on the surface of the cormel is washed, the konjac microsclerotum is soaked in 25 mg / L SA for 1 h, the microsclerotum is divided into multiple microsclerotums along the connection of the cormel, the size and morphology of the cormel are counted, the cormel seeds are sowed after being air-dried for 6 h in a ventilated place, the cormel seeds are sowed in the substrate with a volume ratio of nutrient soil: vermiculite: perlite being 6:3:1, the burying depth is greater than 1 cm, the germination rate is counted after 1 month, and the room temperature is 20-25 ℃ with normal day and night light.

[0062] Glucomannan content detection: the microsclerotum harvested in step S4 is washed to remove the surface medium, cut into small pieces with a size of 0.5-0.8 cm, dried in a blast drying oven at 60 ℃ until constant weight, ground into powder with a mortar, and sieved through an 80-mesh sieve to obtain konjac powder. The konjac powder is added to a 0.1 mol / L formic acid-sodium hydroxide buffer solution with pH 3.0-3.2, shaken in a constant-temperature shaking bed at 25 ℃ for 4 h, and centrifuged in a low-temperature centrifuge at 5000 r / min for 15 min after the konjac powder is fully swelled. The supernatant is the glucomannan extract. The specific steps for determining the glucomannan content in the extract by DNS colorimetry refer to the industry standard NY / T494-2010.

[0063] Example 1

[0064] like Figure 1 The images, from left to right, show konjac tissue culture seedlings, konjac sprouted corms, konjac rhizomes, liquid culture microcorms 1, and liquid culture microcorms 2, all from Example 1. A method for cultivating konjac seeds to increase glucomannan content includes the following steps:

[0065] S1. Regeneration of konjac seedlings: After rinsing the konjac tubers with running water for 1 hour, disinfect them with 75% alcohol for 15 seconds, then disinfect them with 0.1% mercuric chloride for 12 minutes. Finally, rinse them four times with sterile water, cut them into small pieces, and inoculate them onto the germination medium with the buds facing upwards. The germination medium formula is: MS + 0.5% konjac flour + 2.8% sucrose + 2.0 mg / L 6-BA + 0.2 mg / L NAA + 0.2 mg / L LTDZ + 0.15% agar powder + 0.05% activated carbon, pH 5.9. The culture conditions include a light intensity of 1500 lx, a light duration of 12 h / d, with a temperature of 23-25℃ during light and 20-22℃ during darkness, and a culture period of 30 days. The optimal ratio of 6-BA to TDZ in this culture medium formula results in the formation of light green buds on the surface of the tubers on days 7-8 after inoculation. A large number of adventitious buds grow after 15 days, with a germination rate of 100%. On average, each tuber can induce 4.10 ± 0.25 seedlings. Microbulbs are obtained when the base of the regenerated seedlings swells.

[0066] S2. Induction of Konjac Rhizomes: After approximately 25-30 days of culture in step S1, the corms with buds were divided, with the cuts made as clean as possible, and transferred to rhizome induction medium. The rhizome induction medium formula is: modified MS (NO3) - / NH4 + The ratio was 5:1) + 1.5 mg / L 6-BA + 0.2 mg / L NAA + 0.2 mg / L KT + 0.5% agar powder + 2% sucrose + 0.5% mannose + 0.2% mannitol, pH 5.9. Culture conditions: light intensity 1000 lx, light duration 8 h / d, temperature 25℃ during light and 22℃ during darkness. On days 8-10 of culture, the part of the bulb cut surface in contact with the culture medium began to swell, forming white nodular tissue. Multiple fleshy fibrous roots emerged near the buds of the bulb. After 25 days, robust, pinkish-white rhizomes formed, with an average of 4-5 rhizomes induced from each bulb cut.

[0067] S3, regeneration of konjac microsclerotum: after 25 days of S2 culture, the rhizome growing on the sclerotum was cut, 1 / 2 of the aerial roots were cut off with scissors, the fleshy root was reserved, and the cut piece was inoculated into liquid medium (microsclerotum induction medium) for suspension culture to induce microsclerotum formation. The microsclerotum induction medium formula is: MS + 1% mannose + 2% sucrose + 0.3% mannitol + 2.0 mg / L 6-BA + 0.2 mg / L NAA + 0.2 mg / L ZT + 1.0 mg / L arginine, pH 5.8; the culture conditions include 100 r / min shaking speed, 20-22℃ in darkness. When the rhizome is cut, the rhizome with a small amount of root grows well, and after 20 days of suspension culture, many white and greenish new sclerotia are formed.

[0068] S4, microsclerotum proliferation: by combining the sclerotum morphology with section staining observation (safranin-fast green staining), the part of the konjac sclerotum containing somatic embryos is cut off and transferred to the microsclerotum proliferation medium for continuous culture. The microsclerotum proliferation medium formula is modified B5 (adding 0.33 g / L NH4NO3) + 1% mannose + 3% sucrose + 0.2% mannitol + 1.0 mg / L 6-BA + 1.0 mg / L NAA + 0.2 mg / L TDZ + 1.0 mg / L glycine + 1.0 mg / L arginine, pH 5.9, and the culture conditions include light intensity 1000 lx, light time 8 h / d, light quality conditions red light 80-85% + blue light 15-20%, and temperature: 25℃ under light and 22℃ in darkness. Under this condition, the sclerotum has high proliferation efficiency, the proliferation coefficient can reach more than 10 times, and the proliferated sclerotum all has bud points, the average sclerotum diameter reaches more than 1 cm, and the fresh weight reaches more than 5 g.

[0069] Konjac is a herbaceous plant with high light energy utilization rate, and only one leaf is needed for field planting to meet the needs of sclerotum nutrient accumulation. Konjac sclerotum cultured in MS medium has a clear tendency to grow into a cluster of buds, and the sclerotum will consume a large amount of stored substances during the bud and seedling stage, reducing the sclerotum quality. The sclerotum cultured in B5 medium is not easy to grow into seedlings, and B5 medium can be selected as the basic medium for microsclerotum proliferation. However, after several subculture, the sclerotum growing in B5 medium is prone to seedling burn after transplanting due to the unsuitable nitrogen source ratio, so NH4NO3 is added to improve the NH4 + content in B5 medium, thereby improving the nitrogen source ratio in B5 medium, inhibiting the seedling tendency of microsclerotum during the proliferation period, and not affecting the photosynthesis of seedlings, which can better achieve the preparation of excellent seeds.

[0070] S5. Seedling Transplanting: After harvesting, gently scrape off the browned outer skin of the microcorms with scissors, then wash away any remaining culture medium. Soak the konjac microcorms in 25 mg / L SA for 1 hour, then divide them into multiple microcorms along the connecting points. Observe the size and shape of the corms, and air-dry them in a well-ventilated area for 6 hours before sowing. Sow the corm seeds in a substrate with a volume ratio of nutrient soil:vermiculite:perlite of 6:3:1, burying them at a depth greater than 1 cm. Water every other day. Observe the germination rate after one month. Maintain a room temperature of 20-25℃ and normal day and night light.

[0071] Example 2

[0072] like Figure 2 The images, from left to right, show the original microcortex seed cultured in Example 2, the proliferated microcortex, konjac microcortex seeds, konjac seedlings after germination, and complete plants one month after sowing. A method for cultivating konjac seeds to increase glucomannan content includes the following steps:

[0073] S1. Regeneration of Konjac Seedlings: After rinsing the konjac corms with running water for 1 hour, disinfect them with 75% alcohol for 12 seconds, rinse three times with sterile water, then disinfect them with 0.1% mercuric chloride for 12 minutes, and rinse three more times with sterile water. Inoculate the corms with the buds facing upwards onto a sprouting medium. The sprouting medium formula is: MS + 0.55% konjac flour + 3% sucrose + 2.0 mg / L 6-BA + 0.2 mg / L NAA + 0.3 mg / L TDZ + 1.5% agar powder + 0.05% activated carbon, pH 5.9. Culture conditions include a light intensity of 1500 lx, a light duration of 16 h / d, with a temperature of 24℃ during light and 20℃ during darkness, and a culture period of 30 days.

[0074] Adding a certain proportion of activated carbon to the culture medium can block some of the light from the bottom of the medium, which is beneficial to root differentiation.

[0075] S2. Induction of Konjac Rhizomes: After approximately 30 days of culture in S1, the budded corms were cut into small pieces and transferred to rhizome induction medium. The rhizome induction medium formula was: modified MS (NO3- ... 3- / NH4 + The ratio was 3:1) + 1.5 mg / L 6-BA + 0.5 mg / L NAA + 0.2 mg / L KT + 0.5% agar powder + 2% sucrose + 0.5% mannose + 0.2% mannitol, pH 5.9. Culture conditions: light intensity 1000 lx, 12 h / d; temperature: 25℃ under light, 22℃ in darkness.

[0076] S3, regeneration of konjac microsclerotic: after 25 days of culture in S2, the base of the bud appeared swollen nodule callus and split fleshy root-like stems, after longitudinal section, transferred to liquid medium for suspension culture, medium formula: MS + 0.5% mannose + 3% sucrose + 0.3% mannitol + 2.0 mg / L 6-BA + 0.2 mg / L NAA + 0.2 mg / L ZT, 1.0 mg / L arginine, pH 5.8; the culture conditions include shaking speed 90 r / min, temperature 22-24℃.

[0077] S4, proliferation of konjac microsclerotia: when the sclerotia are more than 0.5 cm, the newly formed sclerotia are cut along the swollen base and inoculated into the microsclerotia proliferation medium, the bud point is reserved during inoculation, the sclerotia are half-buried in the microsclerotia proliferation medium, the microsclerotia proliferation medium formula is modified B5 (adding 0.33 g / L NH4NO3) + 0.25% sucrose + 1% mannose + 0.1% mannitol + 1.0 mg / L 6-BA + 1.0 mg / L NAA + 0.3 mg / L TDZ + 0.5% agar powder + 1.0 mg / L glycine + 1.0 mg / L arginine, pH 5.9. The culture conditions include light intensity 1000 lx, light time 8 h / d, light quality conditions red light 80-85% + blue light 15-20%, temperature: 25℃ during light, 22℃ during darkness.

[0078] Konjac prefers diffuse light and weak light, and 1000 lx of light intensity is selected during proliferation culture to avoid damage to the microsclerotia. The microsclerotia induced to form are white and greenish in color within 10 days, and the color of the epidermis changes to green or pink after 20 days of continuous proliferation culture, at which time the sclerotia are in the vigorous growth stage. Figure 3 The results of the section show that the microsclerotia are formed by the proliferation of embryonic cells, and the swollen sclerotia organs have storage function. Calcium oxalate crystals exist in the heteromorphic cells containing glucomannan, and the cytoplasm of the remaining cells is expelled to the edge.

[0079] S5, seedling transplanting: after 28 days of proliferation culture in S4, the sclerotia are taken out of the culture bottle, and the medium remaining on the surface of the sclerotia is washed off. The konjac microsclerotia are soaked in 25 mg / L SA for 1 h, then divided into multiple microsclerotia along the connection of the sclerotia, the size and shape of the sclerotia are counted, and the sclerotia seeds are sown after air-drying for 6 h in a ventilated place. The microsclerotia with seedlings and roots are transplanted after hardening, watered every other day, and the germination rate is counted after 1 month. The room temperature is 20-25℃, and normal day and night light is maintained.

[0080] Example 3

[0081] A konjac seed culture method for increasing the content of glucomannan, comprising the following steps:

[0082] S1, regeneration of Amorphophallus konjac seedling: the flower Amorphophallus konjac tuber is washed with running water for 30 min, then sterilized with 75% alcohol for 15 s, washed with sterile water for 3 times, then sterilized with 0.1% mercury for 12 min, washed with sterile water for 3 times, then the bud point of the tuber is inoculated on the bud culture medium with the bud point upward, the bud culture medium formula: MS + 0.4% Amorphophallus konjac powder + 3% sucrose + 2.0 mg / L 6-BA + 0.2 mg / L NAA + 0.4 mg / L TDZ + 0.15% agar powder + 0.05% activated carbon, pH 5.9. The culture conditions include light intensity 1500 lx, light time 16 h / d, wherein the temperature is 25°C during light and 22°C during darkness, and the culture is performed for 30 d.

[0083] S2, induction of Amorphophallus konjac rhizome: when the culture is performed for about 30 d, the corm with bud is divided into small pieces and transferred to the rhizome induction medium, the rhizome induction medium formula is: modified MS (NO 3- / NH4 + ratio 3:1) + 1.5 mg / L 6-BA + 0.2 mg / L NAA + 0.3 mg / L KT + 0.5% agar powder + 2% sucrose + 1% mannose + 0.1% mannitol, pH 5.9. The culture conditions include light intensity 1000 lx, light time 12 h / d, and temperature: 25°C during light and 22°C during darkness.

[0084] The rhizome cultured for 25 d is subjected to cell staining section. The observation result is shown in Figure 3 (left 2), and the cortex of the rhizome has a large number of thin-walled cells arranged in order, and the nucleolus is obvious, which indicates that it has a vigorous division capacity.

[0085] S3, regeneration of Amorphophallus konjac microsclerotia: after the microsclerotia and the fleshy rhizome are cultured for 25 d in S2, they are divided into pieces in the longitudinal direction and then transferred to the liquid culture medium for suspension culture, the culture medium formula is: MS + 1% mannose + 2% sucrose + 0.3% mannitol + 2.0 mg / L 6-BA + 0.2 mg / L NAA + 0.2 mg / L ZT + 1.0 mg / L arginine, pH 5.8; the culture conditions include shaking speed 100 r / min and temperature 20-22°C.

[0086] S4, the microsclerotia proliferation of konjac: when the swollen sclerotia in S3 exceed 0.5 cm, select the sclerotia with compact structure, white or pink color, and a small amount of adventitious roots when being divided, inoculate into the microsclerotia proliferation medium, and the microsclerotia proliferation medium is modified B5 (0.33 g / L NH4NO3 is added) + 1% mannose + 2% sucrose + 0.2% mannitol + 1.0 mg / L 6-BA + 1.0 mg / L NAA + 0.2 mg / L TDZ + 1.0 mg / L glycine + 1.0 mg / L arginine, pH 5.9. The culture conditions include light intensity 1000 lx, light time 12 h / d, and natural light quality. The temperature is 24 ℃ during light and 22 ℃ during darkness.

[0087] The addition of mannose in the microsclerotia proliferation medium can promote the rapid swelling of the sclerotia and shorten the maturation time. According to the osmotic pressure, the addition of 2% sucrose + 1% mannose is similar to the addition of 4% sucrose, and the growth of konjac inoculated under this condition is good, the microsclerotia swelling coefficient is high, and a plurality of tender green small sclerotia can be formed on the surface of the konjac cuttings at 20 d.

[0088] S5, after the harvested microsclerotia are dropped with residual medium, the microsclerotia are soaked with 25 mg / L SA for 1 h, then are divided into a plurality of microsclerotia along the connecting part of the sclerotia, the size and morphology of the sclerotia are counted, the sclerotia seeds are sowed after being air-dried for 6 h in a ventilated place, the sclerotia seeds are sowed in the medium with a ratio of nutrient soil:vermiculite:perlite being 6:3:1, water is poured once every two days, and the germination rate is counted after 1 month. The room temperature is 20-25 ℃, and normal day and night light is kept.

[0089] As shown in Figure 3 , it is the safranin-fast green staining of the microsclerotia section of konjac. From left to right, they are 200 times micrographs of the center of the sclerotia, the connecting part of the sclerotia and the rootstock cortex. As shown in Figure 3 (left 1) and Figure 3 (left 2), it can be seen that there are a large number of small, cytoplasm-dense, nearly round parenchyma cells in the microsclerotia of konjac, which are prone to form bud eyes in the process of proliferation, so that a large number of detoxified konjac seeds can be prepared through the sclerotium regeneration method. As shown in Figure 3 (left 3), it can be seen that there are a large number of embryonic cells with obvious nucleoli arranged in order under the rootstock cortex of konjac, which have strong proliferation ability.

[0090] The KGM content of the microsclerotia cultured by the application is determined by the DNS colorimetric method according to the industry standard NY / T494-2010, the influence of the mannose addition amount in the microsclerotia proliferation medium on the KGM content, fresh weight and dry weight of the microsclerotia is counted, and the obtained results are as follows, see Table 1.

[0091] Table 1 Influence of mannose addition amount in the proliferation medium on the microsclerotia

[0092] Amount of mannose added / g Inoculum / g Average microsclerotia grain number Fresh weight / g Dry weight / g Glucomannan content 0 3 38 20.8 2.41 29.81％ 5 3 62 28.4 3.66 36.38％ 10 3 80 34.5 4.62 40.02％ 15 3 66 31.8 4.19 39.12％

[0093] The optimal microsclerotum proliferation medium formula of the present application is: modified B5 (add 0.33 g / L NH4NO3) + 1% mannose + 2% sucrose + 0.2% mannitol + 1.0 mg / L 6-BA + 1.0 mg / L NAA + 0.2 mg / L TDZ + 1.0 mg / L glycine + 1.0 mg / L arginine, pH 5.9. Under this condition,

[0094] Comparative Example 1

[0095] Comparative Example 1 adopts the method of inducing microsclerotum from callus provided by Chinese patent CN 113287520A, and the specific steps are as follows:

[0096] Referring to the method of Example 1, the explants of Amorphophallus konjac tubers are detoxified, transferred to adventitious bud medium for induction of adventitious buds, and the adventitious bud induction medium formula is as follows: MS + 1.0 mg / L 6-BA + 0.1 mg / L NAA + 3% sucrose + 0.5% agar powder, pH 5.9, then cut into small pieces and transferred to callus induction medium for dark culture to form callus, the callus induction medium formula is: MS + 0.8 mg / L 6-BA + 0.2 mg / L NAA + 1.0 mg / L 2,4-D + 3% sucrose + 0.5% agar powder, pH 5.9, then transferred to callus proliferation medium for proliferation culture, the callus proliferation medium formula is MS + 1.0 mg / L 6-BA + 0.5 mg / L NAA + 3% sucrose + 0.5% agar powder, and finally transferred to microsclerotum induction medium for induction of microsclerotum in dark environment, the microsclerotum induction medium formula is MS + 0.5 mg / L IBA + 0.5 mg / L NAA + 5% sugar + 0.3% activated carbon. Under this condition, the induction rate of callus-induced microsclerotum is between 70%-80%, and the callus browning rate is about 30%. The total time of this culture route is 90d.

[0097] Referring to the method of Example 1, the rhizomes are induced by using the adventitious buds of Amorphophallus konjac, and the rhizome induction medium formula is: MS + 1.0 mg / L 6-BA + 0.5 mg / L NAA + 3% sucrose + 0.5% agar powder, pH 5.8, and cultured under light intensity of 1000lx for 30d. Under this condition, the rhizome induction rate is 65%, and the morphology is relatively slender.

[0098] Comparative Example 2

[0099] Comparative Example 2 adopts the method of establishing aseptic seedling system provided by Chinese patent CN 113197095A, and the specific steps are as follows:

[0100] The explant of Amorphophallus konjac tuber was detoxified according to the method of Example 1, and was transferred to the adventitious bud culture medium, and the adventitious bud culture medium formula was as follows: MS+1.0 mg / L 6-BA+0.2 mg / L NAA+3% sugar+0.5% agar powder, pH was 5.9. After the adventitious bud was differentiated from the konjac cutting, the konjac seedling was formed after 30 d of continuous culture, and the microsclerotium was formed at the base of the seedling after 90 d of culture.

[0101] The microsclerotium cultured in Example 1 and Comparative Examples 1-2 of the application was used to determine the glucosan content by DNS colorimetry according to the industry standard NY / T494-2010, and the results are shown in Table 2.

[0102] Table 2 Comparison of the efficiency of the microsclerotium prepared by the method of Example 1 of the application and other culture methods

[0103]

[0104] According to the results, the growth cycle of the konjac microsclerotium seed prepared by the method of the application is significantly shortened compared with the prior art, the glucosan content in the microsclerotium is significantly higher than that of the seed prepared by other technologies, and the germination rate after sowing can reach 100%, which can be used as the original seed of konjac planting industry. Applied to production, it can shorten the production cycle and greatly reduce the production cost of konjac seed, and is expected to solve the problem of uneven germplasm resources and yield reduction in konjac planting industry.

[0105] The induction of the konjac rhizome, the regeneration and proliferation culture of the konjac microsclerotium of Example 1-3 and the corresponding results of Comparative Example 1 were statistically analyzed, and the results are as follows, see Table 3.

[0106] Table 3 Statistical results of Example 1-3

[0107] Example Rootstock induction rate Microsclerotia induction rate Proliferation coefficient Average fresh weight of the spheroblasts / g Germination rate Browning rate Example 1 95％ 98％ 10.8 1.09±0.05 100％ 0％ Example 2 93％ 96％ 10.4 1.05±0.04 100％ 1％ Example 3 94％ 96％ 10.5 1.04±0.02 100％ 1％ Comparative Example 1 65％ 77％ 7.2 0.62±0.03 96％ 23％

[0108] From the data in Table 3, it can be seen that the induction rate of the rhizome, the induction rate of the microsclerotium and the proliferation coefficient of the application all reach a high level, the induction rate of the microsclerotium is all above 96%, and the germination rate can all reach 100%, which is significantly better than the prior art, and has a significant progress.

[0109] The plant tissue culture technology and the konjac cell suspension culture system are combined in the application, the konjac bulb is used to induce the rhizome, the microsclerotium of konjac is induced from the parenchyma cells of the rhizome cortex, and a large amount of konjac microsclerotium seed with high glucosan content is obtained through the proliferation culture medium. The microsclerotium obtained by the application has vigorous vitality, strong germination ability, is easy to store, has no dormancy phenomenon, and can be sown at any time, and has a wide application prospect.

[0110] The above merely describes preferred specific embodiments of the present application, but the protection scope of the present application is not limited thereto, and any person skilled in the art, according to the technical solution and inventive concept of the present application, makes equivalent replacement or change within the technical range disclosed by the present application, should be covered within the protection scope of the present application.

Claims

1. A method for cultivating konjac seeds to increase glucomannan content, characterized in that, Includes the following steps: S1. Regeneration of konjac seedlings: Konjac tubers were inoculated into a sprouting medium to induce adventitious buds; the sprouting medium consisted of: 1 / 2-1 MS, 0.4-0.6% konjac flour, 2-3% sucrose, 2.0 mg / L 6-BA, 0.2 mg / L NAA, 0.2-0.5 mg / L TTDZ, 0.1-0.2% agar powder, 0.05% activated carbon, pH 5.8-6.0; S2. Induction of Konjac Rhizomes: The adventitious buds and their bases induced in S1 were separated from the corms, and the separated adventitious buds and their bases were then transferred to a rhizome induction medium to induce the bases of the adventitious buds to swell and form large rhizomes. The rhizome induction medium consisted of: modified MS, 0.5-1.0% mannose, 1-3% sucrose, 0.1-0.3% mannitol, 1.0-2.0 mg / L 6-BA, 0.2-1.0 mg / L NAA, 0.1-0.3 mg / L KT, 0.4-0.6% agar powder, pH 5.8-6.

0. The modified MS was prepared by removing NO3 from the MS medium. - With NH4 + The molar ratio was adjusted to (3-5):1 to obtain the result; S3. Regeneration of konjac microcorals: The large rhizomes and adventitious buds induced in S2 were separated along the swollen area, and half of the aerial roots differentiated near the buds were removed. The rhizomes and corms were then cut into small pieces and transferred to a microcoral induction medium for liquid suspension culture to induce konjac microcorals. The microcoral induction medium consisted of: MS, 0.5-1.0% mannose, 2-3% sucrose, 0.1-0.3% mannitol, 2.0 mg / L 6-BA, 0.2 mg / L NAA, 0.1-0.5 mg / L ZT, 1.0 mg / L arginine, pH 5.8-6.

0. S4. Proliferation culture of konjac microcorals: After making wounds on the epidermis of the konjac microcorals induced in S3, with the buds facing upwards, they were partially embedded in the microcoral proliferation medium to promote the proliferation of a large number of konjac microcorals. The microcoral proliferation medium consisted of: modified B5, 0.5-2.0% mannose, 2-3% sucrose, 0.1-0.2% konjac flour, 0.1-0.3% mannitol, 1.0 mg / L 6-BA, 1.0 mg / L NAA, 0.1-0.5 mg / L TDZ, 0.3-0.5% agar powder, 1.0 mg / L glycine, and 1.0 mg / L arginine, pH 5.8-6.

0. The modified B5 was obtained by adding 0.33 g / L NH4NO3 to B5 medium. S5. Seedling emergence and transplanting: The konjac microcorals proliferated in S4 were soaked in 25 mg / L SA for 1 hour, then divided into multiple microcoral seeds and sown in a substrate with a volume ratio of nutrient soil:vermiculite:perlite of 6:3:1 for 1 month.

2. The method for cultivating konjac seeds to increase glucomannan content according to claim 1, characterized in that, The culture conditions for step S1 include a light intensity of 1500 lx, a light duration of 12 h / d, a temperature of 23-25℃ during light exposure, a temperature of 20-22℃ during darkness, a culture period of 25-30 days, and a pH of 5.

9.

3. The method for cultivating konjac seeds to increase glucomannan content according to claim 1, characterized in that, The culture conditions for step S2 include a light intensity of 1000 lx, a light duration of 8 h / d, a temperature of 23-25℃ during light exposure, a temperature of 20-22℃ during darkness, a culture period of 25-30 days, and a pH of 5.

9.

4. The method for cultivating konjac seeds to increase glucomannan content according to claim 1, characterized in that, Step S3 involves liquid suspension culture in a dark environment.

5. The method for cultivating konjac seeds to increase glucomannan content according to claim 4, characterized in that, The culture conditions for step S3 include a shaking speed of 100 rpm, a dark culture temperature of 20-22℃, a culture time of 15-20 days, and a pH of 5.

8.

6. The method for cultivating konjac seeds to increase glucomannan content according to claim 1, characterized in that, The culture conditions in step S4 include a light intensity of 1000 lx, a light duration of 12 h / d, light quality of 80-85% red light + 15-20% blue light, a temperature of 25°C during light exposure and 22°C during darkness, a culture period of 25-30 days, and a pH of 5.

9.

7. The method for cultivating konjac seeds to increase glucomannan content according to claim 1, characterized in that, Step S2: Cut the bulb into pieces and partially bury them in the rhizome induction medium, with the buds facing upwards.

8. The method for cultivating konjac seeds to increase glucomannan content according to claim 1, characterized in that, When the konjac microcortex induced in step S4 exceeds 0.6 cm in size, the new konjac microcortex is separated from the original konjac microcortex along the connection point, and the new konjac microcortex is inoculated into a microcortex proliferation medium with a thickness of 1.5-2 cm for continued proliferation culture.

9. The method for cultivating konjac seeds to increase glucomannan content according to any one of claims 1 to 8, characterized in that, After soaking for 1 hour in step S5, the connected konjac microbulbs are divided into multiple individual konjac microbulbs and then sown in the substrate.

10. The method for cultivating konjac seeds to increase glucomannan content according to claim 1, characterized in that, The temperature for seedling transplanting and cultivation in step S5 is 20-25℃, and the light condition is natural light.

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