Konjac seed culture method for increasing glucomannan content

Abstract

The invention discloses a konjak seed culture method capable of increasing the content of glucomannan. The konjak seed culture method comprises the following specific steps: S1, regeneration of konjak seedlings; s2, inducing the konjak rhizomes; s3, regeneration of the konjac micro-corms; s4, carrying out multiplication culture on the konjak micro-corms; s5, seedling emergence and transplanting. According to the method, a plant tissue culture technology is combined with a konjak cell suspension culture system, konjak corms are used for inducing rhizomes, konjak micro-corms are induced through parenchyma cells of the cortex of the rhizomes, and then a large number of konjak micro-corm seeds with the high glucomannan content are obtained through a proliferation culture medium. The obtained microcorms are vigorous, high in germination capacity, convenient to store, free of dormancy and capable of being sown at any time, and have wide application prospects.

Description

Technical Field

[0001] The invention relates to the technical field of rapid propagation of medicinal and edible plants, and more specifically to a konjac seed cultivation method for increasing the content of glucomannan. Background Art

[0002] Konjac, also known as konjac, is a perennial herbaceous plant of the genus Amorphophallus Blume in the family Araceae. It can be used as both medicine and food. Its underground corm is its economic organ, and konjac glucomannan (KGM) is its physiologically active main body. Konjac is the only major economic crop in nature that can provide a large amount of glucomannan, and the market demand is large. In recent years, the health function of konjac has been recognized and listed as one of the top ten health foods by the World Health Organization. Studies have found that konjac has physiological effects such as weight loss, lowering blood sugar, lowering blood lipids, resisting inflammation, improving colon mucosal function, and enhancing immunity.

[0003] In production, traditional planting mostly uses corms and rhizomes for asexual reproduction. Due to the problems of konjac bulbs being prone to degeneration, low reproduction coefficient, and damage from pests and diseases, the output of konjac has been declining year by year and cannot meet market demand. Therefore, plant tissue culture technology can be used to study and produce high-quality konjac seedlings with the help of konjac microcorms, and to find a faster and more convenient way of reproduction in tissue culture.

[0004] Konjac microcorms are a kind of micro-modified reproductive organ, which can obtain seed taro through embryonic cell proliferation, achieve the effect of complete virus-free, and reduce the problems of germplasm degradation, yield reduction, quality decline caused by vegetative propagation. Microcorms have the advantages of small size, easy storage and transportation, easy seedling growth and rooting, and are expected to replace test tube seedlings in the propagation of konjac seed taro.

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

[0006] In view of this, the object of the present invention is to provide a rapid propagation system for konjac regenerated microcorms, inducing microcorms through rhizomes, and then obtaining a large number of konjac microcorm seeds with high glucomannan content through corm proliferation, so as to achieve the preparation of excellent original seeds.

[0007] In order to achieve the above object, the present invention is implemented by the following technical solutions:

[0008] A konjac seed cultivation method for increasing the content of glucomannan comprises the following steps:

[0009] S1. Regeneration of konjac seedlings: Konjac tubers were inoculated into budding medium to induce adventitious buds; the budding medium was: 1 / 2-1MS, 0.4-0.6% konjac powder, 2-3% sucrose, 2.0mg / L 6-BA, 0.2mg / L NAA, 0.2-0.5mg / LTDZ, 0.1-0.2% agar powder, 0.05% activated carbon, pH 5.8-6.0;

[0010] S2. Induction of konjac rhizomes: The adventitious buds and the bases of the adventitious buds induced by S1 were cut from the corms, and then the cut adventitious buds and their bases were transferred to the rhizome induction medium to induce the bases of the adventitious buds to swell and form thick rhizomes; the rhizome induction medium was: modified MS, 0.5-1.0% mannose, 1-3% sucrose, 0.1-0.3% mannitol, 1.0-2.0mg / L 6-BA, 0.2-1.0mg / L NAA, 0.1-0.3mg / L KT, 0.4-0.6% agar powder, pH 5.8-6.0; the modified MS was to add NO in the MS medium 3 - With NH 4 + The molar ratio of is adjusted to (3-5):1, and the total nitrogen source concentration remains unchanged;

[0011] S3. Regeneration of konjac micro-corms: The thick rhizomes induced by S2 and the base of the adventitious buds are divided along the swollen part, and 1 / 2 of the aerial roots differentiated near the buds are removed, and then the rhizomes and corms are cut into small pieces and transferred to micro-corm induction medium for liquid suspension culture to induce konjac micro-corms; the micro-corm induction medium is: MS, 0.5-1.0% mannose, 2-3% sucrose, 0.1-0.3% mannitol, 2.0mg / L 6-BA, 0.2mg / L NAA, 0.1-0.5mg / L ZT, 1.0mg / L arginine, pH 5.8-6.0;

[0012] S4. Proliferation culture of konjac microcorms: After the konjac microcorms induced by S3 are cut into wounds on the epidermis, the buds are facing upward and half-buried in a microcorm proliferation medium to promote the proliferation of a large number of konjac microcorms; the microcorm proliferation medium is: improved B5, 0.5-2.0% mannose, 2-3% sucrose, 0.1-0.2% konjac powder, 0.1-0.3% mannitol, 1.0mg / L6-BA, 1.0mg / L NAA, 0.1-0.5mg / L TDZ, 0.3-0.5% agar powder, 1.0mg / L glycine, 1.0mg / L arginine, pH 5.8-6.0; the improved B5 is B5 medium with 0.33g / L NH 4 NO 3 Then get it.

[0013] S5. Seedling transplanting: Soak the konjac microcorms proliferated in S4 in 25 mg / L SA for 1 hour, divide them into multiple microcorms along the connecting parts of the corms, count the size and shape of the corms, and sow the corm seeds in a matrix with a volume ratio of 6:3:1 of nutrient soil: vermiculite:perlite. Count the germination rate after 1 month.

[0014] In a preferred embodiment, the culture conditions of step S1 include light intensity of 1500 lx, light time of 12 h / d, temperature of 23-25°C during light, temperature of 20-22°C during darkness, culture for 25-30 days, and pH 5.9.

[0015] In the preferred embodiment, the culture conditions of step S2 include light intensity of 1000 lx, light time of 8 h / d, temperature of 23-25°C during light, temperature of 20-22°C during darkness, culture for 25-30 days, and pH 5.9.

[0016] In a preferred embodiment, step S3 is to perform liquid suspension culture 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° C., a culture time of 15-20 days, and a pH of 5.8.

[0018] Under the preferred embodiment, the culture conditions of step S4 include light intensity of 1000lx, light time of 12h / d, light quality conditions of 80-85% red light + 15-20% blue light, temperature of 25°C during light, temperature of 22°C in darkness, culture for 25-30d, and pH 5.9.

[0019] In a preferred embodiment, in step S2, the bulb pieces are half-buried in a rhizome induction medium with the bud points placed obliquely upward.

[0020] In a preferred embodiment, when the size of the konjac microcorms induced in step S4 exceeds 0.6 cm, the new konjac microcorms are separated from the original konjac microcorms along the connection, and the new konjac microcorms are inoculated in a microcorm proliferation culture medium with a thickness of 1.5-2 cm to continue proliferation culture.

[0021] In the preferred embodiment, for the seedlings and transplanting in step S5, the culture medium remaining on the surface of the bulbs is firstly washed off, and then the bulbs are broken apart along the connecting parts into individual bulbs, and the bulbs are dried in the shade in a ventilated place for 6 hours before sowing.

[0022] Under the preferred embodiment, the temperature for seedling transplanting and cultivation is 20-25°C, and the lighting condition is natural light.

[0023] Furthermore, the present invention provides a method for cultivating konjac seeds for increasing the content of glucomannan, comprising the following steps:

[0024] S1. Regeneration of konjac seedlings: Konjac tubers are inoculated into a sprouting medium and induced to sprout after light culture; the sprouting medium formula is: in 1 / 2-1MS medium, 0.4%-0.6% konjac powder, 2-3% sucrose, 2mg / L 6-BA, 0.2mg / L NAA, 0.2mg / L TDZ, 0.1%-0.2% agar powder, 1-2g mannitol, 0.05% activated carbon are added, and the pH is adjusted to 5.8-6.0 to induce adventitious buds.

[0025] S2. Induction of konjac rhizomes: The adventitious buds induced in S1 and their bases were cut from the corms and transferred to rhizome induction medium to induce the adventitious buds to germinate and form strong rhizomes; the formula of rhizome induction medium is: modified MS (NO 3 - / NH 4 + The molar ratio is 5:1), 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, and the pH is 5.8-6.0.

[0026] Preferably, the corm selected in step S2 is a modified corm formed by stem expansion at the base of the konjac regenerated seedling.

[0027] S3. Regeneration of konjac microcorms: separate the newly formed rhizomes from the original corms along the swollen part, remove 1 / 2 of the aerial roots differentiated from the microcorms, cut the rhizomes and corms into small pieces and transfer them to microcorm induction medium, and carry out liquid suspension culture in a dark environment to obtain konjac microcorm seeds; the formula of the microcorm induction medium is: MS, 0.5%-1% mannose, 2-3% sucrose, 0.1-0.3% mannitol, 2.0mg / L 6-BA, 0.2mg / L NAA, 0.1-0.5mg / L ZT, 0.3%-0.5% agar powder, 1.0mg / L arginine, pH 5.8-6.0.

[0028] S4. Micro-bulb proliferation culture: After the konjac micro-bulbs obtained in step S3 are cut in the epidermis, they are half-buried in micro-bulb proliferation culture medium and cultured under red and blue light conditions to obtain a large number of konjac micro-bulb seeds; the micro-bulb proliferation culture medium formula is: modified B5 (added with 0.33 g / L NH 4 NO 3 ), 0.5%-2.0% mannose, 2-3% sucrose, 0.1-0.3% mannitol, 1.0mg / L 6-BA, 1.0mg / L NAA, 0.1-0.5mg / L TDZ, 0.4%-0.6% agar powder, 1.0mg / L glycine, 1.0mg / L arginine, pH 5.8-6.0.

[0029] Preferably, in step S4, when the diameter of the new corm exceeds 0.6 cm, the new corm is separated from the main corm along the connection between the corms, with the buds facing upwards, and inoculated into the proliferation medium separately. At the same time, the culture 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: Soak the proliferated konjac micro-corms in 25 mg / L SA for 1 hour, divide them into multiple micro-corms along the connecting parts of the corms, count the size and shape of the corms, and sow the corm seeds in a matrix with a volume ratio of 6:3:1 of nutrient soil: vermiculite:perlite. Count the germination rate after 1 month.

[0031] Preferably, in step S5, the culture medium remaining on the surface of the bulbs is first washed off, and then the bulbs are broken apart along the connecting parts into individual bulbs, and the bulbs are dried in a ventilated place for 6 hours before sowing.

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

[0033] 1. The present invention utilizes the biological characteristics of konjac corms, uses cell engineering technology to prepare the rhizomes of konjac, and produces konjac corm seeds through a suspension culture system. Compared with the traditional technology of forming micro-corms by enlarging the stems of konjac seedlings, the breeding efficiency of the new technology is improved by nearly 100 times, which significantly reduces production costs, shortens the growth cycle of corm seeds, and solves the original seed problem in konjac production.

[0034] 2. The seed vigor of the present invention is high, and the stress resistance is strong: the accumulation of glucomannan in the microcorm is promoted by the proliferation culture medium, and the mature bulb seeds are obtained. The seedling transplanting experiment results show that the konjak microcorm prepared by the present invention has a germination rate of up to 100%, and has strong stress resistance, and can be used as the original seed of the konjak planting industry. The follow-up bioreactor can be used to breed the konjak microcorm on a large scale, thereby realizing the preparation of large quantities of detoxified konjak seeds, giving full play to the advantages of easy storage and transportation of the microcorm, easy seedling growth, small volume, and no dormancy period.

[0035] 3, the present invention means flow process is simple, related micro-corm rapid propagation research, explant-adventitious bud-callus-callus proliferation-inducing micro-corm, need to consume nearly 4 months, and be limited by experimental conditions, callus browning rate is up to more than 30%. Explant-rhizome-corm regeneration-corm proliferation in the present invention, cycle is about 2 months, and after obtaining konjak micro-corm, the operation of corm regeneration-corm proliferation can be repeated, reaching a cultivation cycle of every 30d. In addition, the konjak micro-corm prepared is easy to harvest, only needs to break the corm main body gently, drain after cleaning and can sow, compared to cutting and propagation, wound surface is minimal, and is not easily infected with konjak soft rot. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] In order to more clearly illustrate the technical solution of the embodiment of the present invention, the drawings required for use in the embodiment will be briefly introduced below. It is obvious that the drawings in the following description are only embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on the provided drawings without paying creative work.

[0037] Figure 1 It is the konjac effect diagram of each stage of the method of the present invention.

[0038] Figure 2 The invention relates to the konjac micro-corms at various stages before harvest and after sowing.

[0039] Figure 3 It is a safranin fast green staining of the bulb sections and rhizome sections of the present invention. DETAILED DESCRIPTION

[0040] The technical solutions in the embodiments of the present invention are described clearly and completely below. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work should fall within the scope of protection of the present invention.

[0041] A konjac seed cultivation method for increasing the glucomannan content comprises the following steps:

[0042] S1. Regeneration of konjac seedlings: The tubers of konjac were inoculated into medium 1 / 2-1MS, and 0.4%-0.6% konjac powder, 2-3% sucrose, 2.0mg / L 6-BA, 0.2mg / L NAA, 0.2mg / L TDZ, 0.1%-0.2% agar powder, 0.05% activated carbon were added, pH 5.8-6.0, to induce adventitious buds;

[0043] S2. Induction of konjac rhizomes: The adventitious buds and their bases induced in S1 were cut from the corms, and the cut adventitious buds and their bases were transferred to rhizome induction medium to induce the corm cuts to swell and form strong rhizomes. The rhizome induction medium was modified MS (NO 3 - / NH 4 + The molar ratio is 5:1, the total nitrogen source concentration is 60mmol / L), 0.5-1.0% mannose, 1-3% sucrose, 0.1-0.3% mannitol, 1.0-2.0mg / L 6-BA, 0.2-1mg / L NAA, 0.1-0.3mg / L KT, 0.4-0.6% agar powder, pH is 5.8-6.0;

[0044] S3. Regeneration of konjac micro-corms: separate the thick rhizomes formed in S2 from the original corms along the swollen part, remove 1 / 2 of the aerial roots differentiated from the micro-corms, cut the thick rhizomes and proto-corms into small pieces and transfer them to the micro-molar ratio corm culture medium for regeneration of micro-corms, and carry out liquid suspension culture under dark culture conditions to obtain konjac micro-corm seeds. The micro-corm induction culture medium is: MS, 0.5-1.0% mannose, 2-3% sucrose, 0.1-0.3% mannitol, 2.0mg / L 6-BA, 0.2mg / L NAA, 0.1-0.5mg / L ZT, 1.0mg / L arginine, pH 5.8-6.0;

[0045] S4. Proliferation culture of konjac microcorms: After cutting wounds on the epidermis of the konjac microcorms harvested in step S3, half-bury them in microcorm proliferation culture medium with the buds facing upwards, and carry out proliferation culture for 25-30 days under red and blue light conditions to make the corms expand rapidly and obtain a large number of konjac microcorm seeds. The formula of microcorm proliferation culture medium is: modified B5 (added with 0.33g / L NH 4 NO 3 ), 0.5-2.0% mannose, 2-3% sucrose, 0.1-0.2% konjac flour, 0.1-0.3% mannitol, 1.0mg / L6-BA, 1.0mg / L NAA, 0.1-0.5mg / L TDZ, 1.0mg / L glycine, 1.0mg / L arginine, pH 5.8-6.0.

[0046] S5. Seedling transplanting: Soak the proliferated konjac microcorms in 25 mg / L SA for 1 hour, divide them into multiple microcorms along the connecting parts of the corms, count the size and shape of the corms, and sow the microcorm seeds in a matrix with a volume ratio of 6:3:1 of nutrient soil: vermiculite:perlite after drying in the shade. Count the germination rate after 1 month.

[0047] In certain embodiments of the present invention, the culture conditions of step S1 include a light intensity of 1500 lx, a light time of 12 h / d, a temperature of 23-25° C. during light, a temperature of 20-22° C. during darkness, a culture of 25-30 days, and a pH of 5.9.

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

[0049] In certain embodiments of the present invention, when inducing rhizomes in step S2, the microbulbs are inoculated into a rhizome induction medium, half buried in the medium, and the buds are placed obliquely upward.

[0050] In certain embodiments of the present invention, the culture conditions of step S3 include a shaker speed of 100 rpm, a temperature of 20-22° C. in the dark, a culture time of 15-20 days, and a pH of 5.8.

[0051] In certain embodiments of the present invention, the culture conditions of step S4 include a light intensity of 1000 lx, a lighting time of 12 h / d, a light quality condition of 80-85% red light + 15-20% blue light, a temperature of 25°C in light, 22°C in darkness, and a pH of 5.9.

[0052] In certain embodiments of the present invention, when the size of the new bulb exceeds 0.6 cm in step S4, the new bulb is separated from the original bulb along the connection between the bulbs and inoculated into the proliferation culture medium separately. At the same time, the culture medium should be poured to a thickness of 1.5-2 cm to facilitate the formation of a large number of microbulbs.

[0053] In certain embodiments of the present invention, the culture medium formula in step S1 is: MS, 0.5% konjac flour, 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, pH 5.9;

[0054] The present invention utilizes konjac micro-corms as explants and adopts artificial technology to realize in vitro culture. In adventitious bud culture medium, konjac powder is added, and its gelling property and water retention are utilized to configure semi-solid culture medium, so that the polyphenols produced by accelerating the browning of konjac are diffused in the culture medium, and the browning rate of the explant is reduced. In addition, the main component glucomannan of konjac powder can be absorbed and utilized by konjac explants, thereby greatly shortening the formation time of micro-corms.

[0055] In certain embodiments of the present invention, the rhizome induction medium formula in step S2 is: modified MS (NO 3 - / NH 4 + ratio of 5:1, total nitrogen source concentration of 60mmol / L), 1.5mg / L 6-BA, 1.0mg / L NAA, 0.2mg / L KT, 0.5% agar powder, 2% sucrose, 0.5% mannose, 0.2% mannitol, pH 5.9;

[0056] Konjac microcorms are selected as induction explants because the corms, as seed organs, have a rich epidermis of embryonic cells, which can form adventitious buds through the proliferation and differentiation of embryonic cells, and further induce rhizomes without the need for callus proliferation and culture. In addition, there are abundant thin-walled cells under the cortex of konjac rhizomes, which are ideal materials for corm rapid propagation systems.

[0057] In certain embodiments of the present invention, the microbulb induction medium in step S3 is formulated as 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 the pH is 5.8;

[0058] The main storage substance in konjac corms is glucomannan. By adding the precursors of glucomannan synthesis - mannose and mannitol - to the microcorm induction culture medium, it can effectively promote the proliferation and differentiation of embryonic cell clusters, form bulb organs with storage functions, increase the induction rate of microcorms, and then cultivate konjac stock with strong reproduction potential.

[0059] In certain embodiments of the present invention, the proliferation medium formula in step S4 is as follows: modified B5 (added with 0.33 g / L NH 4 NO 3 ), 1% mannose, 3% sucrose, 0.2% konjac flour, 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, pH 5.9;

[0060] Further cultivation in the proliferation medium can promote the accumulation of glucomannan in the microbulbs, causing the volume to expand rapidly. After planting, the quality standard of the original species can be quickly reached, further improving the reproduction capacity of the microbulbs.

[0061] In certain embodiments of the present invention, the specific operation of seedling transplantation in step S5 is: first, use scissors to gently scrape off the browned epidermis on the surface of the micro-bulbs, wash the culture medium remaining on the surface of the bulbs, soak the konjac micro-bulbs in 25 mg / L SA for 1 hour, divide them into multiple micro-bulbs along the connection of the bulbs, count the size and shape of the bulbs, dry them in a ventilated place for 6 hours, and then sow them, sow the bulb seeds in a matrix with a volume ratio of 6:3:1 of nutrient soil: vermiculite: perlite, and bury them at a depth of more than 1 cm. After 1 month, count the germination rate. The room temperature is 20-25°C, and normal day and night light is maintained.

[0062] Glucomannan content detection: After the micro-bulbs of step S4 are harvested, the surface culture medium is washed off, and the micro-bulbs are cut into small pieces of 0.5cm-0.8cm size, placed in a forced air drying oven and dried to constant weight at 60°C, ground into powder with a medicine mortar, and sieved through an 80-mesh sieve to obtain konjac meal. Konjac meal is added to a formic acid-sodium hydroxide buffer solution of pH 3.0-3.2 of 0.1mol / L, and vibrated in a 25°C constant temperature shaking table for 4h, and after konjac flour is fully swollen, it is centrifuged in a low-temperature centrifuge at 5000r / min for 15min, and the supernatant is taken as the glucomannan extract. The specific steps of using the DNS colorimetric method to measure the glucomannan content in the extract refer to the industry standard NY / T494-2010.

[0063] Example 1

[0064] like Figure 1From left to right are the konjac tissue culture seedlings, konjac sprout corms, konjac rhizomes, liquid cultured micro-corms 1, and liquid cultured micro-corms 2 cultured in Example 1. A method for cultivating konjac seeds with increased glucomannan content comprises the following steps:

[0065] S1. Regeneration of konjac seedlings: After washing the flower konjac tubers with running water for 1 hour, disinfect them with 75% alcohol for 15 seconds, disinfect them with 0.1% mercuric chloride for 12 minutes, and finally wash them with sterile water for 4 times, cut them into small pieces, and inoculate them on bud-growing medium with the bud point facing up. The bud-growing medium formula is: MS+0.5% konjac powder+2.8% sucrose+2.0mg / L6-BA+0.2mg / L NAA+0.2mg / LTDZ+0.15% agar powder+0.05% activated carbon, pH 5.9. The culture conditions include light intensity of 1500lx, light duration of 12h / d, wherein the temperature is 23-25°C during light, and the temperature is 20-22°C during darkness, and the culture is 30d. The formula culture medium contains 6-BA and TDZ in the best ratio. Light green buds are formed on the tuber surface on the 7th to 8th day after inoculation. A large number of adventitious buds grow out after 15 days, with a germination rate of 100%. On average, 4.10±0.25 seedlings can be induced from each tuber. Microbulbs are obtained when the base of the regenerated seedlings swells.

[0066] S2. Induction of konjac rhizomes: When the culture was carried out for about 25-30 days in step S1, the corms with buds were cut, the cuts were as smooth as possible, and the corms were transferred to the rhizome induction medium. The formula of the rhizome induction medium was: modified MS (NO 3 - / NH 4 + The ratio is 5:1) + 1.5mg / L 6-BA + 0.2mg / L NAA + 0.2mg / L KT + 0.5% agar powder + 2% sucrose + 0.5% mannose + 0.2% mannitol, pH 5.9, culture conditions: light intensity 1000lx, light duration 8h / d, light temperature 25℃, dark temperature 22℃. On the 8th to 10th day of culture, the part where the corm cut surface contacts the culture medium begins to swell and form white tumor-like tissue, and multiple fleshy fibrous roots grow near the buds of the corms. After 25 days, thick pink and white rhizomes can be formed. On average, each corm cut can be induced into 4-5 rhizomes.

[0067] S3, regeneration of konjac micro-corms: After 25 days of culture in S2, the rhizomes growing on the corms were divided, 1 / 2 of the aerial roots were cut off with scissors, and the fleshy fibrous roots were retained. After cutting, the pieces were inoculated into liquid culture medium (micro-corm induction medium) for suspension culture to induce micro-corm formation. The formula of micro-corm induction medium was: MS + 1% mannose + 2% sucrose + 0.3% mannitol + 2.0mg / L 6-BA + 0.2mg / L NAA + 0.2mg / L ZT, 1.0mg / L arginine, pH 5.8; the culture conditions included a shaking speed of 100r / min and a temperature of 20-22°C in the dark. When the rhizomes were divided, the rhizomes carrying a small amount of fibrous roots grew well, and many new white green corms were formed after 20 days of suspension culture.

[0068] S4. Microbulb proliferation: Based on the observation of bulb morphology and section staining (safranine fast green staining), the enlarged part of the konjac bulb containing somatic embryos was cut off and transferred to the microbulb proliferation medium for further culture. The microbulb proliferation medium formula was modified B5 (added with 0.33 g / L NH 4 NO 3 )+1% mannose+3% sucrose+0.2% mannitol+1.0mg / L 6-BA+1.0mg / L NAA+0.2mg / L TDZ+1.0mg / L glycine+1.0mg / L arginine, pH 5.9, culture conditions include light intensity 1000lx, lighting time 8h / d, light quality conditions of red light 80-85% + blue light 15-20%, temperature: 25°C in light, 22°C in darkness, under this condition, the bulb proliferation efficiency is high, the proliferation coefficient can reach more than 10 times, and the bulbs after proliferation all have buds, the average bulb diameter reaches more than 1cm, and the fresh weight reaches more than 5g.

[0069] Konjac is a herb with a high light energy utilization rate. When planted in the field, only one leaf is needed to meet the needs of the bulb to accumulate nutrients. Konjac corms cultured in MS medium have an obvious tendency to grow clustered buds. However, during the germination and seedling period, a large amount of storage substances in the corms will be consumed, reducing the quality of the corms. Corms cultured in B5 medium are not easy to produce seedlings. B5 medium can be used as the basic medium for the proliferation of micro-corms. However, after several subcultures, the corms grown in B5 medium are prone to seedling burn after transplanting due to the inappropriate nitrogen source ratio. Therefore, by adding NH 4 NO 3 To increase the NH 4 + content, thereby improving the nitrogen source ratio in the B5 culture medium, thereby inhibiting the tendency of microbulbs to grow seedlings during the proliferation period without affecting the photosynthesis of the seedlings, and better realizing the preparation of high-quality seeds.

[0070] S5. Seedling transplanting: After harvesting, use scissors to gently scrape off the browned epidermis on the surface of the micro-bulbs, and then wash the culture medium remaining on the surface of the bulbs. Soak the konjac micro-bulbs in 25 mg / L SA for 1 hour, and then divide them into multiple micro-bulbs along the connection of the bulbs. Count the size and shape of the bulbs, dry them in a ventilated place for 6 hours, and then sow them. Sow the bulb seeds in a matrix with a volume ratio of 6:3:1 of nutrient soil: vermiculite: perlite, with a burial depth greater than 1 cm, water them every other day, and count the germination rate after 1 month. The room temperature is 20-25℃, and normal day and night light is maintained.

[0071] Example 2

[0072] like Figure 2 From left to right are the original microcorms cultured in Example 2, the microcorms cultured in proliferation, the konjac microcorm seeds, the konjac sprouted seedlings, and the complete plants after one month of sowing. A method for cultivating konjac seeds with increased glucomannan content comprises the following steps:

[0073] S1. Regeneration of konjac seedlings: After washing the flower konjac corm with running water for 1 hour, disinfect it with 75% alcohol for 12 seconds, rinse it with sterile water 3 times, disinfect it with 0.1% mercuric chloride for 12 minutes, and then rinse it with sterile water 3 times, and then inoculate the corm bud point upward on the bud culture medium, the bud culture medium formula is: MS+0.55% konjac powder+3% sucrose+2.0mg / L 6-BA+0.2mg / L NAA+0.3mg / L TDZ+1.5% agar powder+0.05% activated carbon, pH 5.9. The culture conditions include light intensity of 1500lx, light time of 16h / d, temperature of 24°C during light, temperature of 20°C during darkness, and culture for 30d.

[0074] By adding a certain proportion of activated carbon to the culture medium, part of the light at the bottom of the culture medium can be blocked, which is beneficial to root differentiation.

[0075] S2. Induction of konjac rhizomes: After about 30 days of culture in S1, the corms with buds were cut into small pieces and transferred to rhizome induction medium. The formula of rhizome induction medium was: modified MS (NO 3 - / NH 4 + The ratio is 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, 12h / d temperature: 25°C in light, 22°C in dark.

[0076] S3. Regeneration of konjac microcorms: After culturing for 25 days in S2, enlarged tumor-like callus and divided fleshy rhizomes appeared at the base of the buds. After longitudinal cutting, they were transferred to liquid culture medium for suspension culture. The culture medium formula was: 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; culture conditions included a shaking speed of 90 r / min and a temperature of 22-24°C.

[0077] S4. Proliferation of konjac microcorms: When the corms are more than 0.5 cm, cut the new corms along the expanded base and inoculate them into microcorm proliferation medium. Keep the buds during inoculation and half bury the corms into the microcorm proliferation medium. The microcorm proliferation medium formula is modified B5 (added with 0.33 g / L NH 4 NO 3 )+0.25% sucrose+1% mannose+0.1% mannitol+1.0mg / L 6-BA+1.0mg / L NAA+0.3mg / L TDZ+0.5% agar powder+1.0mg / L glycine+1.0mg / L arginine, pH 5.9. The culture conditions include light intensity of 1000lx, light duration of 8h / d, light quality conditions of 80-85% red light+15-20% blue light, temperature: 25°C in light, 22°C in darkness.

[0078] Konjac prefers diffuse light and weak light. When multiplying and culturing, choose a light intensity of 1000lx to avoid strong light from damaging the micro-bulbs. The epidermis of the induced micro-bulbs turns white and green within 10 days. After continuing to multiply and culture for 20 days, the bulbs are in a vigorous growth stage, and the epidermis turns green or pink. Figure 3 The results of the sections showed that the microbulbs expanded to form bulbous organs with storage functions through the proliferation of embryonic cells. Calcium oxalate crystals were present in the heteromorphic cells containing glucomannan, and the cytoplasm of the remaining cells was squeezed to the edge.

[0079] S5, seedling transplanting: After 28 days of proliferation culture in S4, the bulbs were taken out from the tissue culture bottle, and the culture medium remaining on the surface of the bulbs was washed. After soaking the konjac micro-bulbs with 25 mg / L SA for 1 hour, they were divided into multiple micro-bulbs along the connection of the bulbs, and the size and shape of the bulbs were counted. After drying in the shade for 6 hours in a ventilated place, the bulb seeds were sown in a matrix with a volume ratio of 7:2:1 of nutrient soil: vermiculite: perlite. The micro-bulbs that emerged and rooted were hardened and transplanted, watered once every other day, and the germination rate was counted after 1 month. The room temperature was 20-25°C, and normal day and night light was maintained.

[0080] Example 3

[0081] A konjac seed cultivation method for increasing the glucomannan content comprises the following steps:

[0082] S1. Regeneration of konjac seedlings: After washing the flower konjac tubers with running water for 30 minutes, disinfect them with 75% alcohol for 15 seconds, rinse them with sterile water for 3 times, disinfect them with 0.1% mercuric chloride for 12 minutes, rinse them with sterile water for 3 times, and then inoculate the tuber buds upward on the budding medium, the budding medium formula is: MS+0.4% konjac powder+3% sucrose+2.0mg / L 6-BA+0.2mg / L NAA+0.4mg / L TDZ+0.15% agar powder, 0.05% activated carbon, pH 5.9. The culture conditions include light intensity of 1500lx, light time of 16h / d, temperature of 25°C during light, temperature of 22°C during darkness, and culture for 30d.

[0083] S2. Induction of konjac rhizomes: After about 30 days of culture, the bulbs with buds were cut into small pieces and transferred to rhizome induction medium. The formula of rhizome induction medium was: modified MS (NO 3 - / NH 4 + The ratio is 3:1) + 1.5mg / L 6-BA + 0.2mg / L NAA + 0.3mg / L KT + 0.5% agar powder + 2% sucrose + 1% mannose + 0.1% mannitol, pH 5.9. Culture conditions: light intensity 1000lx, light time 12h / d, temperature:

[0084] 25℃ in light and 22℃ in dark.

[0085] After 25 days of culture, the rhizomes were stained and sectioned. Figure 3 As shown in (2nd from left), the cortical part of the rhizome has a large number of neatly arranged thin-walled cells with obvious nucleoli, indicating that it has a strong ability to divide.

[0086] S3. Regeneration of konjac microcorms: After culturing in S2 for 25 days, the microcorms and fleshy rhizomes were cut into pieces longitudinally and transferred to liquid culture medium for suspension culture. The culture medium formula was: 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 included a shaking speed of 100 r / min and a temperature of 20-22°C.

[0087] S4. Proliferation of konjac microcorms: When the corms expanded in S3 exceeded 0.5 cm, select the white or pink corms with dense structure and a small amount of adventitious roots when divided and inoculate them into microcorm proliferation medium. The microcorm proliferation medium formula is modified B5 (added with 0.33 g / L NH 4 NO 3 )+1% mannose+2% sucrose+0.2% mannitol+1.0mg / L 6-BA+1.0mg / L NAA+0.2mg / L TDZ+1.0mg / L glycine+1.0mg / L arginine, pH 5.9. The culture conditions include light intensity of 1000lx, light duration of 12h / d, and light quality conditions of natural light. Temperature: 24°C in light, 22°C in darkness.

[0088] Adding mannose to the microcorm proliferation medium can promote the rapid expansion of the corms and shorten the maturation time. Referring to the osmotic pressure, adding 2% sucrose + 1% mannose is similar to adding 4% sucrose. Under this condition, the inoculated konjac grows well, the microcorm expansion coefficient is high, and multiple tender green small corms can be formed on the surface of the konjac cut pieces at 20 days.

[0089] S5. After removing the residual culture medium from the harvested microbulbs with clean water, soak the microbulbs in 25 mg / L SA for 1 hour, break them into multiple microbulbs along the connecting parts of the bulbs, count the size and shape of the bulbs, dry them in a ventilated place for 6 hours, and sow them. Sow the bulb seeds in a matrix with a ratio of 6:3:1 of nutrient soil: vermiculite:perlite, water them every other day, and count the germination rate after 1 month. The room temperature is 20-25℃, and the light is maintained at normal day and night.

[0090] like Figure 3 Shown is a 200x microscope photo of a konjac microcorm section stained with safranin and fast green. From left to right are the center of the corm, the corm connection, and the rhizome cortex. Figure 3 (Left 1) and Figure 3 (Left 2) It can be seen that there are a large number of small, nearly round, thin-walled cells with dense cytoplasm in the konjac microcorms, which are very easy to form buds during the proliferation process. Therefore, the preparation of large quantities of virus-free konjac seeds can be achieved through corm regeneration. Figure 3 (Left 3) It can be seen that there are a large number of neatly arranged embryonic cells with obvious nucleoli under the cortex of the konjac rhizome, which have strong proliferation ability.

[0091] The microbulbs cultured in the present invention were tested for glucomannan content by DNS colorimetry according to industry standard NY / T494-2010, and the effect of mannose addition in the microbulb proliferation medium on KGM content, fresh weight and dry weight of the microbulbs was statistically analyzed. The results are as follows, see Table 1.

[0092] Table 1 Effect of mannose addition in proliferation medium on microbulbs

[0093] Mannose added amount / g Inoculum amount / g Average number of microbulbs 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％

[0094] The optimal microbulb proliferation medium formula of the present invention is as follows: modified B5 (added with 0.33 g / L NH 4 NO 3 )+1% mannose+2% sucrose+0.2% mannitol+1.0mg / L 6-BA+1.0mg / L NAA+0.2mg / L TDZ+1.0mg / L glycine+1.0mg / L arginine, pH 5.9. Under this condition,

[0095] Comparative Example 1

[0096] Comparative Example 1 adopts the method for callus induction of microbulbs provided by Chinese Patent CN 113287520 A, and the specific steps are as follows:

[0097] Referring to the method of Example 1, the flower konjac tuber was detoxified and transferred to an adventitious bud culture medium for induction of adventitious buds. The adventitious bud induction culture medium was formulated as follows: MS + 1.0 mg / L 6-BA + 0.1 mg / L NAA + 3% sucrose + 0.5% agar powder, pH 5.9, and then cut into small pieces and transferred to a callus induction culture medium for dark culture to form callus. The callus induction culture medium was formulated as follows: 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, and then transferred to a callus proliferation medium for proliferation culture. The callus proliferation culture medium was formulated as MS + 1.0 mg / L 6-BA + 0.5 mg / L NAA + 3% sucrose + 0.5% agar powder, and finally transferred to the microbulb induction medium for microbulb induction in a dark environment. The microbulb induction medium formula is MS + 0.5mg / LIBA + 0.5mg / L NAA + 5% sugar + 0.3% activated carbon. Under this condition, the induction rate of callus induction microbulbs is between 70% and 80%, and the callus browning rate is about 30%. The total time of this culture approach is 90 days.

[0098] With reference to the method of Example 1, the adventitious buds of Amorphophallus konjac were utilized to induce rhizomes. The rhizome induction medium formula was: MS+1.0mg / L 6-BA+0.5mg / L NAA+3% sucrose+0.5% agar powder, pH was 5.8, and cultured for 30d under a light intensity of 1000lx. Under this condition, the rhizome induction rate was 65%, and the morphology was relatively elongated.

[0099] Comparative Example 2

[0100] Comparative Example 2 adopts the method for establishing a sterile seedling system provided by Chinese Patent CN 113197095 A, and the specific steps are as follows:

[0101] With reference to the method of Example 1, the flower konjac tuber is detoxified for explants and transferred to an adventitious bud culture medium, wherein the adventitious bud culture medium is formulated as follows: MS+1.0mg / L6-BA+0.2mg / LNAA+3% sugar+0.5% agar powder, and the pH value is 5.9. After the konjac cuttings differentiate adventitious buds, they are cultured for 30 days to form konjac seedlings. When the konjac cuttings are cultured for 90 days, the stems at the base of the seedlings swell to form micro-bulbs.

[0102] The glucomannan content of the microbulbs cultured in Example 1 of the present invention and Comparative Examples 1-2 was determined by DNS colorimetry according to the industry standard NY / T494-2010. The results are shown in Table 2.

[0103] Table 2 Comparison of the efficiency of preparing microbulbs in Example 1 of the present invention and other culture methods

[0104]

[0105] As can be seen from the results, the konjac micro-corm seeds prepared by the inventive method have a significantly shortened growth cycle compared to the prior art, the glucomannan content in the micro-corm is significantly higher than the seeds prepared by other technologies, and the germination rate after sowing can reach 100%, which can be used as the original seed of the konjac planting industry. Application in production can shorten the production cycle, greatly reduce the production cost of konjac seeds, and is expected to solve the problem of uneven germplasm resources and reduced yield in the konjac planting industry.

[0106] The results of induction of konjac rhizomes, regeneration and proliferation culture of konjac microcorms in Examples 1-3 and the corresponding results of Comparative Example 1 were statistically analyzed, and the results obtained are as follows, see Table 3.

[0107] Table 3 Statistical results of Examples 1-3

[0108] Implementation Cases Rhizome induction rate Microbulb induction rate Proliferation coefficient Average fresh weight of bulbs / 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％

[0109] It can be seen from the data in Table 3 that the rhizome induction rate, microbulb induction rate and proliferation coefficient of the present invention all reach a relatively high level, the microbulb induction rate is all above 96%, and the germination rate can reach 100%, which is significantly better than the prior art and represents a significant improvement.

[0110] The present invention combines plant tissue culture technology with a konjac cell suspension culture system, utilizes konjac corms to induce rhizomes, induces konjac microcorms through thin-walled cells in the cortex of the rhizomes, and then obtains a large number of konjac microcorm seeds with high glucomannan content through a proliferation culture medium. The microcorms obtained by the present invention are vigorous, have strong germination ability, are easy to store, have no dormancy, can be sown at any time, and have broad application prospects.

[0111] The above description is only a preferred specific implementation manner of the present invention, but the protection scope of the present invention is not limited thereto. Any technician familiar with the technical field can make equivalent replacements or changes according to the technical scheme and inventive concept of the present invention within the technical scope disclosed by the present invention, which should be covered by the protection scope of the present invention.

Claims

1. A konjac seed culture method for increasing the content of glucomannan, characterized in that: The steps include: S1. Regeneration of konjac seedlings: The konjac tubers were inoculated into a budding medium to induce adventitious buds; the budding medium was: 1 / 2-1MS, 0.4-0.6% konjac powder, 2-3% sucrose, 2.0mg / L 6-BA, 0.2mg / L NAA, 0.2-0.5mg / LTDZ, 0.1-0.2% agar powder, 0.05% activated carbon, pH 5.8-6.0; S2. Induction of konjac rhizomes: The adventitious buds and the bases of the adventitious buds induced by S1 are cut from the corm, and then the cut adventitious buds and their bases are transferred to the rhizome induction medium to induce the bases of the adventitious buds to swell and form thick rhizomes; the rhizome induction medium is: modified MS, 0.5-1.0% mannose, 1-3% sucrose, 0.1-0.3% mannitol, 1.0-2.0mg / L 6-BA, 0.2-1.0mg / L NAA, 0.1-0.3mg / L KT, 0.4-0.6% agar powder, pH 5.8-6.0; the modified MS is to add NO3 - With NH4 + The molar ratio of is adjusted to (3-5):1 to obtain; S3. Regeneration of konjac micro-corms: The thick rhizomes induced by S2 and the base of the adventitious buds are divided along the swollen part, and 1 / 2 of the aerial roots differentiated near the buds are removed, and then the rhizomes and corms are cut into small pieces and transferred to micro-corm induction medium for liquid suspension culture to induce konjac micro-corms; the micro-corm induction medium is: MS, 0.5-1.0% mannose, 2-3% sucrose, 0.1-0.3% mannitol, 2.0mg / L6-BA, 0.2mg / L NAA, 0.1-0.5mg / L ZT, 1.0mg / L arginine, pH 5.8-6.0; S4. Proliferation culture of konjac microcorms: After a wound is cut in the epidermis of the konjac microcorms induced by S3, the buds are facing upward and half-buried in a microcorm proliferation medium to promote the proliferation of a large number of konjac microcorms; the microcorm proliferation medium is: improved B5, 0.5-2.0% mannose, 2-3% sucrose, 0.1-0.2% konjac powder, 0.1-0.3% mannitol, 1.0mg / L6-BA, 1.0mg / L NAA, 0.1-0.5mg / L TDZ, 0.3-0.5% agar powder, 1.0mg / L glycine, 1.0mg / L arginine, pH 5.8-6.0; the improved B5 is obtained by adding 0.33g / L NH4NO3 to the B5 medium. S5. Seedling transplanting: The konjac microcorms proliferated and cultured in S4 were soaked in 25 mg / L SA for 1 hour, divided into multiple microcorm seeds, and sown in a matrix with a volume ratio of 6:3:1 of nutrient soil: vermiculite:perlite for 1 month.

2. The konjac seed culture method for improving glucomannan content according to claim 1, wherein The culture conditions of step S1 include light intensity of 1500 lx, light time of 12 h / d, temperature of 23-25° C. during light, temperature of 20-22° C. during darkness, culture for 25-30 days, and pH 5.

9.

3. The konjac seed culture method for improving glucomannan content according to claim 1, wherein The culture conditions of step S2 include light intensity of 1000 lx, light time of 8 h / d, temperature of 23-25° C. during light, temperature of 20-22° C. during darkness, culture for 25-30 days, and pH 5.

9.

4. The konjac seed culture method for improving glucomannan content according to claim 1, wherein Step S3 performs liquid suspension culture in a dark environment.

5. The konjac seed culture method for improving glucomannan content according to claim 4, wherein: The culture conditions of step S3 include a shaking speed of 100 rpm, a dark culture temperature of 20-22° C., a culture time of 15-20 days, and a pH of 5.

8.

6. The konjac seed culture method for improving glucomannan content according to claim 1, wherein: The culture conditions of step S4 include light intensity of 1000 lx, light time of 12h / d, light quality conditions of 80-85% red light + 15-20% blue light, temperature of 25°C during light, temperature of 22°C in darkness, culture for 25-30d, and pH 5.

9.

7. The konjac seed culture method for improving glucomannan content according to claim 1, characterized in that: Step S2: half-bury the bulb cut pieces in rhizome induction medium, with the bud points placed obliquely upward.

8. The konjac seed culture method for improving glucomannan content according to claim 1, characterized in that: When the size of the konjac microcorms induced in step S4 exceeds 0.6 cm, the new konjac microcorms are separated from the original konjac microcorms along the connection, and the new konjac microcorms are inoculated in a microcorm proliferation culture medium with a thickness of 1.5-2 cm to continue proliferation culture.

9. The method for cultivating konjac seeds for increasing the content of glucomannan according to any one of claims 1 to 8, characterized in that: Step S5: After soaking for 1 hour, the connected konjac micro-corms are divided into a plurality of individual konjac micro-corms and then sown in the matrix.

10. The konjac seed culture method for increasing the glucomannan content according to claim 1, characterized in that: The temperature for seedling transplanting and cultivation in step S5 is 20-25° C., and the lighting condition is natural lighting.

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