CN116267606B - Rapid propagation method of konjak seeds - Google Patents
Rapid propagation method of konjak seeds Download PDFInfo
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- CN116267606B CN116267606B CN202310174401.8A CN202310174401A CN116267606B CN 116267606 B CN116267606 B CN 116267606B CN 202310174401 A CN202310174401 A CN 202310174401A CN 116267606 B CN116267606 B CN 116267606B
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Classifications
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
- A01H4/00—Plant reproduction by tissue culture techniques ; Tissue culture techniques therefor
- A01H4/008—Methods for regeneration to complete plants
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
- A01H4/00—Plant reproduction by tissue culture techniques ; Tissue culture techniques therefor
- A01H4/002—Culture media for tissue culture
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- Life Sciences & Earth Sciences (AREA)
- Developmental Biology & Embryology (AREA)
- Engineering & Computer Science (AREA)
- Biotechnology (AREA)
- Cell Biology (AREA)
- Botany (AREA)
- Environmental Sciences (AREA)
- Breeding Of Plants And Reproduction By Means Of Culturing (AREA)
Abstract
The invention provides a propagation method of konjak seeds, which comprises the following steps: s1, inducing aseptic seedlings of konjak; s2, inducing propagation stems of konjak; s3, propagation of propagation stems; s4, inducing konjak microsphere stems, and performing physiological maturation treatment on the konjak microsphere stems. The konjak seeds prepared by the method disclosed by the invention are smaller in volume, convenient to transport and store, and capable of obtaining the konjak seeds through embryogenic cell proliferation, realizing a complete detoxification effect, and reducing the problems of germplasm degradation, yield reduction, quality reduction and the like caused by nutrition proliferation; meanwhile, the seeding machine has no dormancy phenomenon, can sow and germinate at any time, does not need complicated operations such as hardening off, and has the effects of shortening the production period and reducing the cost.
Description
Technical Field
The invention relates to the field of crop cultivation, in particular to a asexual propagation method of konjak reproductive stem seeds.
Background
Konjak is called konjak, and is a perennial herb of the genus konjak (Amorphopha llus Blume) of the family Araceae. The harvesting part is underground expansion corm, which is plant organ with highest known glucomannan (Konjac Glucomannan, KGM) content, and also contains polysaccharides, alkaloids, vitamins, mineral elements, and nutrients such as various amino acids, ceramide, flavonoids, etc. The KGM is soluble hemicellulose and has various unique physicochemical properties of water solubility, water retention thickening, stability, suspension, gelation, adhesion, film formation and the like. KGM has important application value in the fields of functional foods, medicines, chemical industry, petroleum exploitation, environmental protection and the like, and particularly has very wide application in the field of health care foods. Konjak has been developed into various functional foods for weight loss, lipid and blood sugar reduction, cancer resistance, aging resistance and the like, and has an important effect on improving the intestinal microenvironment, and has been listed as one of ten health foods by the united nations health organization. The konjak is the only main economic plant capable of providing a large amount of KGM in the nature, has higher medical care value and economic value, and has better application prospect in the fields of green food development, medical care, disease control, chemical industry and the like. With the rapid development of the China health industry, the demand of konjak products is rapidly increased, and the demand of konjak fine powder is more than 20 ten thousand tons every year worldwide. China is one of countries with the largest planting area of konjak in the world, and reaches about 450 mu, more than 150 konjak processing enterprises at the downstream, and 2.5 ten thousand tons of konjak fine powder and micropowder are produced in one year.
But the prominent problems in the konjak planting process are as follows: (1) high seed production costs. The konjak uses mature corms as propagation seeds, about 4000 seeds are needed per mu, about 200kg of seeds are needed per mu on average, the planting cost is very high, and the large planting area and the large seed quantity for production of the konjak all over the country can cause great economic loss only by producing reserved seeds; (2) The traditional konjak planting method is limited by seasons and external environmental conditions, and the problems of slow propagation speed, long growth period and the like of seed propagation exist; (3) Traditional konjak seeds belong to nutritional tubers, and occupy a large amount of space, so that the konjak seeds are not beneficial to storage and transportation; (4) As konjak belongs to nutrition propagation, problems of variety degradation, soft rot hazard, serious sitting obstacle hazard and the like occur along with the extension of planting years, so that the quality of konjak germplasm is reduced; (5) As the virus in the tissue slowly accumulates after the konjak seeds are reserved for many years, the quality is reduced to a certain extent, the yield is also reduced sharply, and the loss reaches more than 30%. The tissue culture mode is adopted for seedling preparation, and although the efficiency is high and the method is not limited by seasons, climates and lands, the method still has some problems: (1) As the number of subcultures increases, hormone accumulation results in weaker seedlings; (2) The labor capacity is large, and the existing tissue culture seedling preparation requires a great deal of manpower, and the time and labor are wasted in transferring and subculturing; (3) Before cultivation, the operation links of seedling hardening, transplanting and the like are needed, and places with strict requirements such as a specific seedling hardening greenhouse and the like are also needed, so that labor cost is increased; (4) is disadvantageous for mechanical production.
Disclosure of Invention
The invention aims to provide a seed for konjak production and a rapid propagation method thereof, which are detoxified, high in yield and low in cost, so as to solve the problems that the konjak seed prepared by the prior art has poor detoxication effect, dormancy phenomenon, incapability of seeding and sprouting at any time, low yield, complicated operations such as seedling hardening and the like, thereby causing overhigh production cost and longer production period.
In order to achieve the above object, the present invention provides a propagation method of konjak seeds, characterized by comprising the steps of:
s1, induction of konjak aseptic seedlings: adding 5-10% corn juice, 0.1-0.5 mg/L6-BA, 0.2-0.7% agar powder, 0.1-0.8g/L konjak coarse powder, pH5.5-6.5 into 1/8-1MS culture medium, and inducing sprouting;
s2, induction of propagation stems of konjak: transferring the bud induced in the S1 into a propagation stem induction culture medium for inducing the occurrence and growth of the propagation stem, wherein the propagation stem induction culture medium is as follows: 1/8-1MS, 0.2-1.0% konjak coarse powder, 0.1-0.7% agar powder, 0.1-0.6mg/L brassinolide, 0.5-1.0 mg/L6-BA, 0.1-1.0g/L K 2 HPO 4 0.1-1.0g/L Ca (NO) 3 ) 2 0.1-1.0mg/L glycine, 0.1% active carbon, 1-6% sucrose, pH5.5-6.5;
s3, propagation of propagation stems: cutting out the stem segments with swelling, and transferring the stem segments to a proliferation culture medium for proliferation culture, wherein the proliferation culture medium is as follows: 1/8-1MS, 0.2-0.5% konjak coarse powder, 0.1-0.7% agar powder, 0.1-1.0mg/L TDZ, 0.1-1.0mg/L zeatin, 0.4-1.0g/L K 2 HPO 4 0.1-1.0g/L Ca (NO) 3 ) 2 0.1-0.5mg/L glycine, 3-6% sucrose, 0.1% active carbon, pH5.5-6.5;
s4, induction of konjak microsphere stems: placing the propagation stems obtained in the step S3 into an induction culture medium for culture,the induction culture medium is as follows: 1/8-1MS, 0.1-0.5% konjak coarse powder, 0.1-1.0mg/L TDZ, 0.1-1.0mg/L zeatin, 0.1-1.0 mg/L6-BA, 0.1-1.0g/L K 2 HPO 4 0.1-1.0g/L KCL, 0.1-0.8g/L Ca (NO) 3 ) 2 3-6% of sucrose, 0.1-0.5mg/L of glycine, 0.1-0.7% of agar powder, 0.1% of active carbon and pH of 5.5-6.5;
s5, physiological maturation treatment of konjak ball seeds: washing the bulb seeds induced in the step S4, treating with 1-5mmol/L oxalic acid solution, treating with 0.1-1.0mg/L ABA solution after washing, washing and draining.
Preferably, the culture conditions of step S1 include an illumination intensity of 30 to 120. Mu.M.m -2 ·s -1 8-12h/d, wherein the temperature is 23-27 ℃ in illumination and 19-21 ℃ in darkness, and the pH is 5.5-6.5.
Preferably, the culture conditions of step S2 include an illumination intensity of 20 to 50. Mu.M.m -2 ·s -1 8-10h/d, wherein the temperature is 22-26 ℃ in illumination and 18-22 ℃ in darkness, and the pH is 5.5-6.5.
Preferably, the culture conditions of step S3 include culturing at a temperature of 19-21℃in the dark at a pH of 5.5-6.5 when the diameter of the propagation stem obtained in step S2 is 1cm or more.
Preferably, the culture conditions of step S4 include a pH of 5.5-6.0 and a dark condition of 22-24 ℃.
Preferably, the medium formulation described in step S1: 1/4MS, 8% corn juice, 0.5 mg/L6-BA, 0.5% agar powder, 0.2% konjak meal; culture conditions: light intensity: 50 mu M M -2 ·s -1 The method comprises the steps of carrying out a first treatment on the surface of the Temperature: light is at 25 ℃ and dark is at 20 ℃.
Preferably, the propagation stem induction medium formulation in step S2: 1/4MS, 0.6% konjak coarse powder, 0.2% agar powder, 0.5mg/L brassinolide, 0.8 mg/L6-BA, 0.5g/L K 2 HPO 4 Ca (NO) 0.5mg/L 3 ) 2 0.6mg/L glycine, 3% sucrose, 0.1% active carbon, pH6.0, culture conditions: the light intensity was 40. Mu.M.m -2 ·s -1 8h/d, when the light is irradiated: 24 ℃, in the dark: 20 ℃.
Preferably, in step S3The proliferation medium formula comprises: 1/2MS, 0.4% konjak meal, 0.6mg/L TDZ, 0.8mg/L zeatin, 0.8g/L K 2 HPO 4 Ca (NO) 0.5g/L 3 ) 2 5% sucrose, 0.3mg/L glycine, 0.1% active carbon, 0.3% agar powder and pH value of 6.0; culture conditions: dark culture, 20 ℃.
Preferably, the medium formulation described in step S4: 1/2MS, 0.4% konjak meal, 0.6mg/L TDZ, 0.6 mg/L6-BA, 0.6mg/L zeatin, 0.5g/L K 2 HPO 4 KCl 0.5g/L, ca (NO) 0.5g/L 3 ) 2 5% sucrose, 0.2mg/L glycine, 0.1% activated carbon, 0.2% agar powder, pH6.0, culture conditions: 23℃under dark conditions.
Preferably, the physiological ripening treatment method of konjak ball seeds in the step S5 comprises the steps of treating the konjak ball seeds with 3mmol/L oxalic acid solution for 3h, taking out clear water, washing 2-3 times, then soaking with 0.5mg/L ABA solution for 8h, washing with clear water, draining off water, and storing at 0-4 ℃.
The invention has the following beneficial effects:
the invention utilizes biological characteristics of konjak plants, prepares test-tube konjak by screening propagation stems and utilizing plant cell engineering means, can utilize a bioreactor to realize large-scale factory breeding of konjak seeds, and solves various problems in the production of konjak seeds. The invention utilizes propagation stems developed in the rapid propagation of konjak to quickly culture in vitro by artificial means, further develops into konjak stock with strong fertility, can realize the production advantages of low cost, short period, no season limitation, detoxification, easy storage and transportation and the like of konjak seed production, greatly promotes the rapid development of konjak seed industry and meets the requirements of konjak planting.
The invention firstly breeds tissue cells with the potential of developing into expansion tubers in stem organs, cuts the tissue cells into embryogenic tissue cell masses after being removed, further breeds the embryogenic tissue cell masses into tissue masses which are provided with a plurality of growth centers and are formed by embryogenic cells, and cuts the tissue masses into small pieces, so that konjak seeds with complete germination capacity can be developed by the method, and the breeds stem breeds, differentiation, expansion, development and maturation are prepared into test tube seed konjak with 100 percent of germination rate and 100 percent of detoxification rate through the stage of cell differentiation determination under the in vitro condition. Compared with the asexual propagation seedling in the prior art, the propagation efficiency is improved by hundreds of times, and the cost is greatly reduced; is a new konjak seed breeding mode; the prepared konjak blocks have the hair percentage reaching 100 percent, have no dormancy phenomenon and are sowed at any time; solving the problem of seed detoxification; can be produced industrially and in large scale, is not limited by seasons, climates, weather and land, and can be used for producing seeds all year round.
Compared with the traditional konjak seeds, the konjak seeds prepared by the method have smaller volume, can reach 0.5 cm with the diameter of less than 1.0cm, and are convenient to transport and store; the seed taro is obtained through the proliferation of embryogenic cells, the objective detoxification effect is realized, the complete detoxification is realized, and the problems of germplasm degradation, yield reduction, quality reduction and the like caused by nutrition propagation are reduced; the seedling culture device has no dormancy phenomenon, can sow and germinate at any time, does not need complicated operations such as hardening off, has the effect of shortening the production period, and saves the dormancy and germination relieving time by at least 2 months; the seed of the konjak can also be used as a seed for liquid culture, the efficiency can reach 5000 test tube seed blocks/L, the seed requirement of konjak planting in 1.5 mu land is met, the seed quantity is 3500-4000 according to the traditional planting, the cost of 1L of the seed prepared by the technology is about 50 yuan, the average seed cost per mu is about 25 yuan, and the seed cost of konjak industry is greatly reduced.
Drawings
FIG. 1 is a diagram showing effects of the method of the present invention at each stage of cultivation of konjak.
Detailed Description
The invention will be further described with reference to specific examples and figures.
The method of the invention mainly adopts the technical route: the culture medium and conditions of each step are further defined by taking nutrition and hormone required by konjak block molding, temperature and illumination into consideration.
S1, preparing aseptic seedlings: sterilizing rhizoma Amorphophalli tuber, cutting into pieces, and adding into 1/8-1 MS: 5-10% corn juice, 01-0.5mg/L of 6-BA (6-benzylaminoadenine), 0.2-0.7% of agar powder, 0.1-0.8g/L of konjak powder (coarse powder), pH of 5.5-6.5, inducing sprouting, and culturing conditions: light irradiation of 30-120 mu M.m -2 ·s -1 8-12h/d, temperature: 25+/-2 ℃ in illumination and 20+/-1 ℃ in darkness;
s2, induction of propagation stems on aseptic seedlings: transferring the aseptic seedlings obtained in the step S1 to a propagation stem induction culture medium, wherein the propagation stem induction culture medium is as follows: 1/8-1MS, 0.2-1.0% konjak powder (coarse powder), 0.1-0.7% agar powder, 0.1-0.6mg/L brassinolide, 0.5-1.0 mg/L6-BA, 0.1-1.0g/L K 2 HPO 4 0.1-1.0g/L Ca (NO) 3 ) 2 0.1-1.0mg/L glycine, 0.1% active carbon, 1-6% sucrose, pH5.5-6.5, culture conditions: illumination of 20-50 mu M.m -2 ·s -1 8-10h/d, temperature: 24+/-2 ℃ in illumination and 20+/-2 ℃ in darkness;
s3, breeding and proliferation of propagation stems: the expanded propagation stem segments containing somatic embryos are cut off by morphological combination of frozen section and microscopic observation and placed on a feed proliferation medium for feed proliferation culture, wherein the feed proliferation medium is as follows: 1/8-1MS, 0.2-0.5% konjak flour (coarse powder), 0.1-0.7% agar powder, 0.1-0.8mg/L TDZ (TDZ, N-phenyl-N' -1,2, 3-thiadiazole-5-urea), 0.1-1.0mg/L zeatin, 0.4-1.0g/L K 2 HPO 4 0.1-1.0g/L Ca (NO) 3 ) 2 0.1-0.5mg/L glycine, 0.1% active carbon, 3-6% sucrose, pH5.5-6.5, culture conditions: dark, 20.+ -. 1 ℃.
S4, breeding stems to induce konjak ball seeds: cutting multiplication stem into small pieces when the diameter of multiplication stem is above 1cm, culturing on seed induction medium, 1/8-1MS, 0.1-0.5% konjak coarse powder, 0.1-1.0mg/L TDZ, 0.1-1.0mg/L zeatin, 0.1-1.0 mg/L6-BA, and 0.1-1.0g/L K 2 HPO 4 0.1-1.0g/L KCl, 0.1-0.8g/L Ca (NO) 3 ) 2 3-6% of sucrose, 0.1-0.5mg/L of glycine, 0.1-0.7% of agar powder, 0.1% of active carbon, pH of 5.5-6.5, and culture conditions: darkness, 23+ -1deg.C; after culturing for 30-40d, 8-10 konjak ball seeds are formed in each proliferation block on average;
s5, a konjak ball seed after-ripening promoting culture medium: taking out the bulb seeds cultured in the step S4, treating 2-4 with 1-5.0mmol/L oxalic acid solution, taking out clear water, washing 2-3 times, then soaking with 0.1-1.0mg/L ABA (abscisic acid) solution for 6-10h, washing with clear water, draining off water, and storing at low temperature of 0-4 ℃ at 8-14 ℃.
Example 1
S1, induction of aseptic seedlings of konjak: washing rhizoma Amorphophalli tuber with running water for 30min, washing with sterile water for 3-4 times, sterilizing with 70-75% alcohol for 15s, and adding H 2 O 2 Sterilizing the solution for 20min, flushing with sterile water for three times, and transferring the sterilized konjak block-cutting bud point upwards into bud differentiation culture medium, wherein the buds: 1/4MS+8% corn juice+0.5 mg/L6-BA+0.5% agar powder+0.2% konjak powder, pH6.0, and inducing sprouting. Culture method and conditions: illumination with 50 mu M.m light intensity -2 ·s -1 10h/d, temperature: light is at 25 ℃ and dark is at 20 ℃. The formula and the culture conditions are optimal, the germination rate is 100%, the buds are strong, and the average bud height is 4+/-1 cm when the induction is carried out for 20 days.
S2, induction of propagation stems of konjak: at about 20-25 days of S1 induction culture, the sprouted tissue is transferred to propagation stem induction medium, the propagation stem induction medium formulation: 1/4MS+0.6% konjak flour (coarse powder) +0.2% agar powder+0.5 mg/L brassinolide+0.8 mg/L6-BA+0.5 g/L K 2 HPO 4 +0.5g/L Ca (NO) 3 ) 2 +0.6mg/L glycine+3% sucrose+0.1% active carbon, pH6.0, culture conditions: the light intensity was 40. Mu.M.m -2 ·s -1 8h/d, when the light is irradiated: 24 ℃, in the dark: 20 ℃. Under the culture medium and the culture conditions, more than 4 propagation stems appear at the base of each bud within the culture time of 10-15 days, and 2 expansion points (propagules with a large number of embryogenic cells) appear on each propagation stem on average, wherein the diameter of each expansion propagation bulb reaches more than 1.5cm after the culture is finished.
S3, propagation of propagation stems: after culturing for 30 days in S2, a large number of stems or roots grow on the basal part contacted with the solid culture medium or below the culture medium, and the stems which are swelled are propagation stems after morphological observation and microscopic examination. Cutting propagation stem segment with large number of meristematic cells to obtainStem segments are subjected to multiplication culture on a new culture medium, wherein the culture medium is a stem segment multiplication culture medium: 1/2MS+0.4% konjak flour (coarse powder) +0.6mg/L TDZ+0.8mg/L zeatin+0.8 g/L K 2 HPO 4 +0.5g/L Ca (NO) 3 ) 2 +5% sucrose+0.3 mg/L glycine+0.1% active carbon+0.3% agar powder, pH value is 6.0, and the culture is performed in darkness at 20 ℃. Under the condition, the propagation efficiency of propagation stems in vitro is high, the multiplication times reach more than 15 times, and the diameter of each propagation stem is more than 2 cm.
S4, inducing konjak microsphere stalk seeds to form by a solid culture medium: cutting the expanded propagation stems of the S3 into small blocks, transferring the small blocks to a new culture medium, and adopting a culture medium formula: 1/2MS+0.4% konjak flour (coarse powder) +0.6mg/L TDZ+0.6 mg/L6-BA+0.6 mg/L zeatin+0.5 g/L K 2 HPO 4 +0.5g/L KCl+0.5g/L Ca (NO) 3 ) 2 +5% sucrose+0.2 mg/L glycine+0.1% active carbon+0.2% agar powder, pH value is 6.0, culture conditions are: 23 ℃ under dark conditions; under the condition, the efficiency of converting the propagation stems into konjak bulbs is high, a large number of konjak bulbs are propagated on each propagation stem cutting block, and 8 konjak bulbs appear on average in each small block.
S5, performing physiological maturation treatment on konjak microsphere stem seeds; taking out the bulb seeds cultured in the step S4, treating the bulb seeds with 3mmol/L oxalic acid solution for 3 hours, taking out clean water, washing the bulb seeds for 2-3 times, then soaking the bulb seeds with 0.5mg/L ABA (abscisic acid) solution for 8 hours, washing the bulb seeds with clean water, draining water, and storing the bulb seeds at a low temperature of 12 ℃.
As shown in figure 1, from top to bottom, the method comprises a sterile seedling stage, a sterile seedling growing propagation stem stage, a propagation stem and root stage, a propagation stem expanding stage, a propagation stem growing into a bulb stage, a propagation stem bulb growing stage, a bulb increasing stage, a bulb growing stage in liquid, a mature microsphere seed diameter less than 1cm stage, a mature microsphere seed stage, a microsphere seed germination stage and a microsphere seed seedling stage.
Example 2
S1, washing konjak tubers with tap water for 20min, washing 3-4 times with sterile water, sterilizing with 73% alcohol for 12s, and then with H 2 O 2 Sterilizing the solution for 30min, and then flushing with sterile waterWashing for three times, and cutting aseptic konjak into pieces with the size: 0.5x0.5x0.5 cm 3 The bud point is upwards transferred to a culture medium, and the culture medium is: 1/8MS+5% corn juice+0.3 mg/L6-BA+0.3% agar powder+0.4% konjak powder, pH6.5, induced sprouting, culture conditions: 40 mu M.m-2.s -1 8h/d, temperature: 24 ℃ under illumination and 19 ℃ under darkness;
s2, transferring the tissue with buds to a new culture medium when the induction culture is carried out for 25 days, wherein the culture medium comprises the following formula: 1/2MS+0.4% konjak flour (coarse powder) +0.4% agar powder+0.3 mg/L brassinolide+0.5 mg/L6-BA+0.3 g/L K 2 HPO 4 +0.8mg/L Ca (NO) 3 ) 2 +0.4mg/L glycine+2% sucrose+0.1% active carbon, pH6.5, culture conditions: 30 mu M M -2 ·s -1 10h/d, when the light is irradiated: 26 ℃, when dark: 21 ℃;
s3, after 30 days of culture, a plurality of stolons appear on the base of the aseptic seedling, the stems with expansion points are cut off to form stem segments, and the stem segments are transferred to a new culture medium, wherein the formula comprises the following steps: 1/4MS+0.3% konjak flour (coarse powder) +0.4mg TDZ/L+0.5mg zeatin+0.5 g/L K 2 HPO 4 +0.3mg/L Ca (NO) 3 ) 2 +3% sucrose+0.5 mg/L glycine+0.1% active carbon+0.6% agar powder, pH5.5; culturing in dark at 19 ℃;
s4, cutting the expanded propagation stems into small blocks, and transferring the small blocks to a konjak bulb block forming culture medium, wherein the formula of the culture medium is as follows: 1/4MS+0.3% konjak flour (coarse powder) +1.0mg/L TDZ+0.8 mg/L6-BA+0.8 mg/L zeatin+1.0 g/L K 2 HPO 4 +0.8g/L KCl+0.8g/L Ca (NO) 3 ) 2 +4% sucrose+0.1 mg/L glycine+0.1% active carbon+0.4% agar powder, pH6.5, culture conditions: 24 ℃ under dark condition; after 30 days of culture, konjak micro-corm seeds with the diameter of about 1cm are developed.
S5, performing physiological maturation treatment on konjak microsphere stem seeds; taking out the bulb seeds cultured in the step S4, treating the bulb seeds with 5mmol/L oxalic acid solution for 4 hours, taking out clean water, washing the bulb seeds for 2-3 times, then soaking the bulb seeds with 1.0mg/L ABA (abscisic acid) solution for 8 hours, washing the bulb seeds with clean water, draining water, and storing the bulb seeds at a low temperature of 10 ℃.
Example 3
S1, establishing a konjak aseptic seedling system: washing Amorphophallus konjac under tap water for 1 hr, sucking off excessive water on the surface with sterile filter paper, sterilizing with 70% alcohol for 10s, soaking in sterile water for 1s, repeating for 3 times, and using 0.1% HgCl 2 Sterilizing the solution for 20min, flushing with sterile water for three times, cutting the sterilized konjak into pieces, and placing the tissue with bud points on a bud promoting culture medium upwards, wherein the culture medium comprises the following formula: 1/2MS+10% corn juice+0.6 mg/L6-BA+0.7% agar powder+0.3% konjak powder, pH5.8, induced sprouting, culture conditions: 80 mu M M -2 ·s -1 18h/d, temperature: 26 ℃ under illumination and 19 ℃ under darkness;
s2, transferring the tissue blocks growing buds to a new culture medium after the induction growth culture is carried out for 30 days, wherein the culture medium comprises the following formula: 1/8MS+0.2% konjak flour (coarse powder) +0.7% agar powder+0.7 mg/L brassinolide+1.0 mg/L6-BA+0.8 g/L K 2 HPO 4 +0.3g/L Ca (NO) 3 ) 2 +0.8mg/L glycine+0.1% active carbon+5% sucrose, pH6.0, culture conditions: 50 mu M M -2 ·s -1 12h/d, temperature: 24 ℃ under illumination and 20 ℃ under darkness;
s3, after the culture for 30 days, a plurality of propagation stems grow on the base part of the aseptic seedling, the stolon with expansion points is cut into stem segments, and the stem segments are transferred to a new culture medium, wherein the formula comprises the following components: 1/8MS+0.5% konjak flour (coarse powder) +0.8mg/LTDZ+1.0mg/L zeatin+1.0 g/L K 2 HPO 4 +0.8g/L Ca(NO 3 ) 2 +6% sucrose+0.2 mg/L glycine+0.1% active carbon+0.2% agar powder, pH6.0. Dark culture at 20deg.C
S4, cutting the continuously-expanding propagation stems into small pieces, transferring the small pieces to a new culture medium, and adopting a culture medium formula: 1/2MS+0.5% konjak flour (coarse powder) +0.8mg/L TDZ+1.0 mg/L6-BA+1.0 mg/L zeatin+0.6 g/L K 2 HPO 4 +0.6g/L KCl+0.3g/L Ca (NO) 3 ) 2 +6% sucrose+0.3 mg/L glycine+0.1% charcoal+0.5% agar powder, pH5.5, culture conditions: 22 ℃ under dark conditions; after 30 days of culture, konjak micro-corm seeds with the diameter of about 1cm are developed.
S5, performing physiological maturation treatment on konjak microsphere stem seeds; taking out the bulb seeds cultured in the step S4, treating the bulb seeds with 1mmol/L oxalic acid solution for 2 hours, taking out clean water, washing the bulb seeds for 2-3 times, then soaking the bulb seeds with 0.1mg/L ABA (abscisic acid) solution for 8 hours, washing the bulb seeds with clean water, draining water, and storing the bulb seeds at a low temperature of 8 ℃.
Example 4
S1, establishing a konjak aseptic seedling system: washing Amorphophallus konjac under tap water for 1 hr, sucking off excessive water on the surface with sterile filter paper, sterilizing with 70% alcohol for 10s, soaking in sterile water for 1s, repeating for 3 times, and using 0.1% HgCl 2 Sterilizing the solution for 20min, flushing with sterile water for three times, cutting the sterilized konjak into pieces, and placing the tissue with bud points on a bud promoting culture medium upwards, wherein the culture medium comprises the following formula: MS+5% corn juice+0.8 mg/L6-BA+0.2% agar powder+0.1% konjak powder, pH5.5, induced sprouting, culture conditions: the light intensity is 100 mu M M -2 ·s -1 8h/d, temperature: 23 ℃ in illumination and 19 ℃ in darkness;
s2, transferring the tissue blocks with the induced growth buds to a new culture medium, wherein the formula of the culture medium comprises the following steps: MS+0.8% konjak flour (coarse powder) +0.1% agar powder+0.2 mg/L brassinolide+0.3 mg/L6-BA+1.0 g/L K 2 HPO 4 +1.0g/L Ca (NO) 3 ) 2 +0.2mg/L glycine, pH6.0, culture conditions: 100 mu M.m -2 ·s -1 10h/d, temperature: 26 ℃ under illumination and 18 ℃ under darkness;
s3, growing a plurality of propagation stems on the base part of the aseptic seedling, cutting the stolons with expansion points into stem segments, and transferring the stem segments into a shake flask containing a liquid culture medium, wherein the formula comprises the following components: MS+0.2% konjak flour (coarse powder) +0.3mg TDZ/L+0.4mg/L zeatin+0.4 g/L K 2 HPO 4 +1.0g/L Ca(NO 3 ) 2 +4% sucrose+0.1 mg/L glycine+0.1% active carbon+0.1% agar powder, pH6.0. Dark culture, 22 ℃.
S4, cutting the continuously-expanding propagation stems into small pieces, transferring the small pieces into a shake flask containing a new liquid culture medium, and placing the shake flask into a shaking table for culture, wherein the formula of the culture medium is as follows: 1/8MS+0.2% konjak flour (coarse powder) +0.2mg/L TDZ+0.4mg/L zeatin+0.4 mg/L6-BA+0.8 g/L K 2 HPO 4 +0.3g/L KCl+0.2g/L Ca (NO) 3 ) 2 +2% sucrose+0.5 mg/L GlycineAcid+0.1% active carbon+0.1% agar powder, pH5.8, culture conditions: 22 ℃ under dark conditions; after 30 days of culture, konjak micro-corm seeds with the diameter of about 1cm are developed.
S5, performing physiological maturation treatment on konjak microsphere stem seeds; taking out the bulb seeds cultured in the step S4, treating the bulb seeds with 2mmol/L oxalic acid solution for 3 hours, taking out clean water, washing the bulb seeds for 2-3 times, then soaking the bulb seeds with 0.1mg/L ABA (abscisic acid) solution for 8 hours, washing the bulb seeds with clean water, draining water, and storing the bulb seeds at a low temperature of 14 ℃.
TABLE 1 proliferation effect and germination Rate of microsphere stock under liquid culture
The optimal scheme of the invention is as follows:
1. the optimal culture medium formula for bud induction comprises the following components: 1/4MS+8% corn juice+0.5 mg/L6-BA+0.5% agar powder+0.2% konjak powder; optimal culture conditions: light intensity: 50 mu M M -2 ·s -1 The method comprises the steps of carrying out a first treatment on the surface of the Temperature: 25 ℃ when the light is irradiated and 20 ℃ when the dark is irradiated;
2. propagation stem optimal induction medium and conditions: 1/4MS+0.6% konjak flour (coarse powder) +0.2% agar powder+0.5 mg/L brassinolide+0.8 mg/L6-BA+0.5 g/L K 2 HPO 4 +0.5mg/L Ca (NO) 3 ) 2 +0.6mg/L glycine+3% sucrose+0.1% active carbon, pH6.0, culture conditions: the light intensity was 40. Mu.M.m -2 ·s -1 8h/d, when the light is irradiated: 24 ℃, in the dark: 20 ℃;
3. the optimal culture medium for breeding stem and stem segment breeding and proliferation: 1/2MS+0.4% konjak flour (coarse powder) +0.6mg/L TDZ+0.8mg/L zeatin+0.8 g/L K 2 HPO 4 +0.5g/L Ca (NO) 3 ) 2 +5% sucrose+0.3 mg/L glycine+0.1% active carbon+0.3% agar powder, pH value is 6.0, and the culture is carried out in darkness at 20 ℃;
4. konjak ball seeds form an optimal culture medium: 1/2MS+0.4% konjak flour (coarse powder) +0.6mg/L TDZ+0.6 mg/L6-BA+0.6 mg/L zeatin+0.5 g/L K 2 HPO 4 +0.5g/L KCl+0.5g/L Ca (NO) 3 ) 2 +5% sucrose+0.2 mg/L glycine+0.1% charcoal+0.2% agar powder, pH6.0, culture conditions: 23 ℃ under dark conditions;
5. the optimal treatment process for post-ripening and germination promotion of konjak ball seeds comprises the following steps: 3mmol/L oxalic acid solution is used for 3h, clear water is taken out for washing 2-3 times, then 0.5mg/L ABA (abscisic acid) solution is used for soaking and treating for 8h, clear water is used for washing, water is drained, and the treatment temperature is 12 ℃ and the storage is carried out at low temperature.
TABLE 2 comparison of the present technique with other methods of preparing konjak seeds
TABLE 3 experiment of germination and seedling formation of post-ripe konjak bulb seeds
| Total number of tests (granule) | Germination number (grain) | Average bud count/grain | Germination percentage% | |
| Konjak bulb seed | 500 | 500 | 3 | 100 |
Compared with the traditional konjak seeds, the konjak seeds prepared by the method have smaller volume, can reach 0.5 cm with the diameter of less than 1.0cm, and are convenient to transport and store; the seed taro is obtained through the proliferation of embryogenic cells, the objective detoxification effect is realized, the complete detoxification is realized, and the problems of germplasm degradation, yield reduction, quality reduction and the like caused by nutrition propagation are reduced; the seedling culture device has no dormancy phenomenon, can timely sow and germinate, does not need complicated operations such as hardening off, has the effect of shortening the production period, and saves the dormancy and germination relieving time by at least 2 months; the seed of the konjak can also be used as a seed for liquid culture, the efficiency can reach 5000 test tube seed blocks/L, the seed requirement of konjak planting in 1.5 mu land is met, the seed quantity is 3500-4000 according to the traditional planting, the cost of 1L of the seed prepared by the technology is about 50 yuan, the average seed cost per mu is about 25 yuan, and the seed cost of konjak industry is greatly reduced.
The foregoing is only a preferred embodiment of the present invention, but the scope of the present invention is not limited thereto, and any person skilled in the art, who is within the scope of the present invention, should be covered by the protection scope of the present invention by making equivalents and modifications to the technical solution and the inventive concept thereof.
Claims (10)
1. The propagation method of konjak seeds is characterized by comprising the following steps:
s1, induction of konjak aseptic seedlings: adding 5-10% corn juice, 0.1-0.5 mg/L6-BA, 0.2-0.7% agar powder, 0.1-0.8g/L konjak coarse powder, pH5.5-6.5 into 1/8-1MS culture medium, and inducing sprouting;
s2, induction of propagation stems of konjak: transferring the bud induced in the S1 into a propagation stem induction culture medium for inducing the occurrence and growth of the propagation stem, wherein the propagation stem induction culture medium is as follows: 1/8-1MS, 0.2-1.0% konjak coarse powder, 0.1-0.7% agar powder, 0.1-0.6mg/L brassinolide, 0.5-1.0 mg/L6-BA, 0.1-1.0g/LK 2 HPO 4 0.1-1.0g/L Ca (NO) 3 ) 2 0.1-1.0mg/L glycine, 0.1% active carbon, 1-6% sucrose, pH5.5-6.5;
s3, propagation of propagation stems: cutting out the stem segments with swelling, and transferring the stem segments to a proliferation culture medium for proliferation culture, wherein the proliferation culture medium is as follows: 1/8-1MS, 0.2-0.5% konjak coarse powder, 0.1-0.7% agar powder, 0.1-1.0mg/L TDZ, 0.1-1.0mg/L zeatin, 0.4-1.0g/L K 2 HPO 4 0.1-1.0g/L Ca (NO) 3 ) 2 0.1-0.5mg/L glycine, 3-6% sucrose, 0.1% active carbon, pH5.5-6.5;
s4, induction of konjak microsphere stems: placing the propagation stems obtained in the step S3 into an induction culture medium for culture, wherein the induction culture medium is as follows: 1/8-1MS, 0.1-0.5% konjak coarse powder, 0.1-1.0mg/L TDZ, 0.1-1.0mg/L zeatin, 0.1-1.0 mg/L6-BA, 0.1-1.0g/L K 2 HPO 4 0.1-1.0g/L KCl, 0.1-0.8g/L Ca (NO) 3 ) 2 3-6% of sucrose, 0.1-0.5mg/L of glycine, 0.1-0.7% of agar powder, 0.1% of active carbon and pH of 5.5-6.5;
s5, physiological maturation treatment of konjak ball seeds: washing the bulb seeds induced in the step S4, treating with 1-5mmol/L oxalic acid solution, treating with 0.1-1.0mg/L ABA solution after washing, washing and draining.
2. The propagation method of konjak seeds according to claim 1, wherein the cultivation condition of step S1 includes an illumination intensity of 30 to 120. Mu.M.m -2 ·s -1 8-12h/d, wherein the temperature is 23-27 ℃ in illumination and 19-21 ℃ in darkness, and the pH is 5.5-6.5.
3. The propagation method of konjak seeds according to claim 1, wherein the cultivation condition of step S2 includes an illumination intensity of 20 to 50. Mu.M.m -2 ·s -1 8-10h/d, wherein the temperature is 22-26 ℃ in illumination and 18-22 ℃ in darkness, and the pH is 5.5-6.5.
4. The propagation method of konjak seeds according to claim 1, wherein the culture condition of step S3 includes culturing at a temperature of 19-21 ℃ at a pH of 5.5-6.5 in the dark when the diameter of the propagation stem section obtained in step S2 is 1cm or more.
5. The propagation method of konjak seeds according to claim 1, wherein the cultivation condition of step S4 includes a pH of 5.5 to 6.0 and a dark condition of 22 to 24 ℃.
6. The propagation method of konjak seeds according to claim 1, wherein the medium formula in step S1: 1/4MS, 8% corn juice, 0.5 mg/L6-BA, 0.5% agar powder, 0.2% konjak meal; culture conditions: light intensity: 50 mu M M -2 ·s -1 The method comprises the steps of carrying out a first treatment on the surface of the Temperature: light is at 25 ℃ and dark is at 20 ℃.
7. The propagation method of konjak seeds according to claim 1, wherein the propagation stem induction medium formula in step S2: 1/4MS, 0.6% konjak coarse powder, 0.2% agar powder, 0.5mg/L brassinolide, 0.8 mg/L6-BA, 0.5g/L K 2 HPO 4 Ca (NO) 0.5g/L 3 ) 2 0.6mg/L glycine, 3% sucrose, 0.1% active carbon, pH6.0, culture conditions: the light intensity was 40. Mu.M.m -2 ·s -1 8h/d, when the light is irradiated: 24 ℃, in the dark: 20 ℃.
8. The propagation method of konjak seeds according to claim 1, wherein the propagation medium formula in step S3: 1/2MS, 0.4% konjak meal, 0.6mg/L TDZ, 0.8mg/L zeatin, 0.8g/L K 2 HPO 4 Ca (NO) 0.5g/L 3 ) 2 5% sucrose, 0.3mg/L glycine, 0.1% active carbon, 0.3% agar powder and pH value of 6.0; culture conditions: dark culture, 20 ℃.
9. The propagation method of konjak seeds according to claim 1, wherein the medium formula in step S4: 1/2MS, 0.4% konjak meal, 0.6mg/L TDZ, 0.6mg/L6-BA, 0.6mg/L zeatin, 0.5g/L K 2 HPO 4 KCl 0.5g/L, ca (NO) 0.5g/L 3 ) 2 5% sucrose, 0.2mg/L glycine, 0.1% activated carbon, 0.2% agar powder, pH6.0, culture conditions: 23℃under dark conditions.
10. The propagation method of konjak seeds according to claim 1, characterized in that the physiological ripening treatment of konjak ball seeds of step S5 comprises treating the bulb seeds with 3mmol/L oxalic acid solution for 3 hours, taking out clear water and washing 2-3 times, then soaking with 0.5mg/L ABA solution for 8 hours, washing with clear water, draining off water, and storing at 0-4 ℃.
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