CN103548593A - Method for promoting Codonopsis pilosula seed germination by utilizing bacteriabeneficial beneficial bacteria - Google Patents
Method for promoting Codonopsis pilosula seed germination by utilizing bacteriabeneficial beneficial bacteria Download PDFInfo
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Abstract
The invention relates to a method for promoting Codonopsis pilosula seed germination by utilizing bacteriabeneficial beneficial bacteria. The method utilizes solution of Bacillus subtilis belonging to bacteriabeneficial growth promotion microorganisms to steep Codonopsis pilosula seeds, therefore, germination potential and germination rate of the Codonopsis pilosula seeds are remarkably increased, vigor and emergence rate of the Codonopsis pilosula seeds are increased, problems of small Codonopsis pilosula seeds, low germination potential and germination rate and irregular emergence during production are solved, and economic losses on labor and material resources caused by seedling supplement and repeated germination of the Codonopsis pilosula are reduced. Furthermore, the Bacillus subtilis has the advantages of being free of toxicity and harm to people and livestock, free of pollution to environment, non-pathogenic and strong in stress resistance, and can produce a variety of growth hormone-like chemicals and rich antimicrobial substances, promote plant growth and improve plant growth and development. The method is simple, can be widely applied to cultivation of the Codonopsis pilosula, and has important social, economic and ecological economic value.
Description
Technical field
The invention belongs to microbiology and plant physiology, relate to and utilize beneficial rhizosphere bacterium bacillus subtilis GB03 bacterial strain to improve Radix Codonopsis potentiality of seed and germination rate, thereby promote the method for Radix Codonopsis seed germination, after assurance planting seed, field emergence is healthy and strong, whole consistent.
Background technology
The features such as that bacillus subtilis (Bacillus subtilis) has is nontoxic, free from environmental pollution to people and animals, non-pathogenic and strong stress resistance, it can produce multiple growth hormone material and abundant antibacterial material, Promoting plant growth, improve vine growth and development, increase the defensive ability/resistance ability of plant to adverse circumstances such as salt stresses, thereby improve the disease resistance of plant.
In recent years, Radix Codonopsis has been obtained new progress at faces such as treatment coronary heart disease, hyperlipemia, hypopiesis, make its consumption speed increase (Ma Xuemei and Wu towards peak 2009), but because of Radix Codonopsis seed little, life-span is short, and during direct sowing and seedling, germination rate is low, and during cultivation, seeding quantity is large, cost is high, does not reach production requirement.According to investigations, the demand of domestic annual Radix Codonopsis is considerably beyond production, from Radix Codonopsis as commodity since city's floor trading, just often there is the phenomenon of big rise and big fall in the production and selling of Radix Codonopsis, price fluctuation is also very large.At present, national Radix Codonopsis wild resource reserves are about 1.58 ten thousand tons, but resource is disperseed, output presents downward trend year by year, and the commodity amount that can provide is extremely limited, and Radix Codonopsis market supply and demand contradiction is outstanding (Liu Dejun etc. 2001) very.From Chinese medicine Radix Codonopsis resource investigation result, show, Radix Codonopsis main product in the market in Shanxi, the province such as Gansu, Shaanxi, Sichuan and Hubei, be mainly cultivation product, a small amount of is wild, Gansu is except original producton location, nearly large area development decades Radix Codonopsis is planted, and has expanded the production scale of plain colored Radix Codonopsis simultaneously, becomes the producing region (Xiangdong Zhang etc. 2013) of Chinese medicine Radix Codonopsis maximum.
At present, along with the enforcement of the modernization of Chinese medicine and the status of China in World Economics more and more higher, all steady growths of the production of traditional Chinese medicine and outlet, the outlet of China's Radix Codonopsis in 2007 has surpassed 0.3 ten thousand tons according to estimates, as herbal pharmaceutical raw material, the also phenomenal growth of the consumption of Radix Codonopsis.In recent years, the repeatedly lifting of the market price of Radix Codonopsis, and because national Radix Codonopsis gross yield obviously declines, its price is significantly raised.Supply falls short of demand has become a great problem of Chinese medicine in producing for codonopsis pilosula, therefore, improve Radix Codonopsis and produce soil quality, regulation activity composition and Metabolite Accumulation, improving Radix Codonopsis quality has become the first-selection of traditional Chinese medicine plant husbandry, is also problem demanding prompt solution during Radix Codonopsis is produced.
Summary of the invention
The object of the invention is to overcome the deficiencies in the prior art, provide a kind of beneficial rhizosphere bacterium to promote the method for Radix Codonopsis seed germination.Be to utilize beneficial rhizosphere bacterium bacillus subtilis GB03 to promote the sprouting of Radix Codonopsis seed, the method is simple to operate, and cost is low, efficient, nontoxic and environmentally friendly harmless.
For achieving the above object, the technical scheme that the present invention takes is: a kind of beneficial rhizosphere bacterium promotes the method for Radix Codonopsis seed germination, and its main feature is that step is:
(1) Radix Codonopsis seed disinfection is processed: the Radix Codonopsis seed of full seed N/D, uniformity, and with after 75% alcohol-pickled 8-10s, use 5% clorox to soak 10-12min, the distilled water flushing of use sterilizing 7-8 time, in 2-4 ℃ of refrigerator overnight;
(2) bacillus subtilis GB03 bacterium solution preparation: first prepare bacteriolyze meat soup LB liquid nutrient medium: be 1:2:1 preparation according to the ratio of sodium chloride, tryptone and dusty yeast, solvent is distilled water; Adjust pH to 7.2, high temperature is 115-132 ℃ of sterilizing, after temperature to be cooled is 20-35 ℃, the LB liquid nutrient medium of every 100mL adds the bacillus subtilis GB03 bacterium liquid of 50-100 μ L, at rotating speed, be under 180-250r/min, temperature is on the constant-temperature shaking incubator of 25-30 ℃ after overnight incubation, and mensuration bacterial concentration is OD
600during for 0.8-1.0, take out standby;
(3) planting daughter bacteria liquid processes: step (1) seed of finishing dealing with is soaked in to 5min-20min in bacillus subtilis GB03 bacterium liquid, then bacterium liquid is poured out, the seed of finishing dealing with is sowed.
Beneficial effect of the present invention: the present invention can significantly improve Radix Codonopsis potentiality of seed and germination rate, thereby improve Radix Codonopsis seed vitality and emergence rate, keep more neat germination rate, overcome the upper Radix Codonopsis seed of production little, germination vigor and germination rate are low, irregular problem of emerging, produces to reduce the economic losses such as human and material resources that upper Radix Codonopsis is filled the gaps with seedlings and repeatedly germinateed and cause.Meanwhile, the feature such as that bacillus subtilis has is nontoxic, free from environmental pollution to people and animals, non-pathogenic and strong stress resistance, can produce multiple growth hormone material and abundant antibacterial material, and Promoting plant growth, improves vine growth and development.Method for treating seeds of the present invention is simple, can be widely used in the cultivation of Radix Codonopsis, has important society, economy and Eco-economic Value.
Accompanying drawing explanation
Fig. 1: sprout Radix Codonopsis seed sprouting situation comparison after 3 days.In figure, four little figure represent respectively contrast, beneficial bacteria bacterium immersion bubble Radix Codonopsis seed 5 minutes, 10 minutes and 20 minutes;
Fig. 2: sprout Radix Codonopsis seed germination seedlings root developmental state comparison after 3 days.The picture left above is beneficial bacteria bacterium immersion bubble Radix Codonopsis seed 5 minutes, and left and right figure is contrast; The Radix Codonopsis germination seed of figure below for amplifying;
Fig. 3: the impact of GB03 bacterium immersion bubble on Radix Codonopsis potentiality of seed.In figure, four pillars represent respectively the Radix Codonopsis potentiality of seed of contrast, beneficial bacteria bacterium immersion bubble Radix Codonopsis seed 5 minutes, 10 minutes and 20 minutes;
Fig. 4: the impact of GB03 bacterium immersion bubble on Radix Codonopsis percentage of seedgermination.In figure, four pillars represent respectively the Radix Codonopsis percentage of seedgermination of contrast, beneficial bacteria bacterium immersion bubble Radix Codonopsis seed 5 minutes, 10 minutes and 20 minutes.
Embodiment
Below principle of the present invention and feature are described, example, only for explaining the present invention, is not intended to limit scope of the present invention.The those of ordinary skill of described technical field, according to above content disclosed by the invention and scope that each parameter is got, all can be realized object of the present invention.
Embodiment 1: a kind of beneficial rhizosphere bacterium promotes the method for Radix Codonopsis seed germination, and its main feature is that step is:
(1) Radix Codonopsis seed disinfection is processed: the Radix Codonopsis seed of full seed N/D, uniformity, and with after 75% alcohol-pickled 8s, use 5% clorox to soak 10min, the distilled water flushing of use sterilizing 7-8 time, in 4 ℃ of refrigerator overnight;
(2) bacillus subtilis GB03 bacterium solution preparation:
First prepare LB liquid nutrient medium: according to sodium chloride 5g/L, the ratio preparation of tryptone 10g/L and dusty yeast 5g/L, with standard type pH meter (PB-10, Beijing Sai Duolisi) adjust pH to 6.8, use vertical pressure steam sterilization pan (LDZX-50KBS, Shen, Shanghai peace) high temperature 118-125 ℃ sterilizing after cooling, the LB liquid nutrient medium of every 100mL adds the GB03 bacterium liquid of 50 μ L, at rotating speed, be under 180r/min, temperature is the constant-temperature shaking incubator (THZ-300 of 28 ℃, the permanent science and technology in Shanghai one) after upper overnight incubation, (NanoDrop 1000 to use detection of nucleic acids instrument, when mensuration bacterial concentration is OD600=0.9, take out standby Germany), bacterium liquid is now with the current.
(3) planting daughter bacteria liquid processes: step (1) seed of finishing dealing with is soaked in to 5min in bacillus subtilis GB03 bacterium liquid, then bacterium liquid is poured out, the seed of finishing dealing with is sowed.
Embodiment 2: a kind of beneficial rhizosphere bacterium promotes the method for Radix Codonopsis seed germination, and its main feature is that step is:
(1) Radix Codonopsis seed disinfection is processed: the Radix Codonopsis seed of full seed N/D, uniformity, and with after 75% alcohol-pickled 9s, use 5% clorox to soak 11min, the distilled water flushing of use sterilizing 7-8 time, in 2 ℃ of refrigerator overnight;
(2) bacillus subtilis GB03 bacterium solution preparation:
First prepare bacteriolyze meat soup LB liquid nutrient medium: according to the ratio of sodium chloride, tryptone and dusty yeast, be 1:2:1 preparation, solvent is distilled water; Adjust pH to 7.2, high temperature is 115-118 ℃ of sterilizing, after temperature to be cooled is 20 ℃, the LB liquid nutrient medium of every 100mL adds the bacillus subtilis GB03 bacterium liquid of 80 μ L, at rotating speed, be under 180-250r/min, temperature is on the constant-temperature shaking incubator of 25 ℃ after overnight incubation, and mensuration bacterial concentration is OD
600during for 0.8-1.0, take out standby.
(3) planting daughter bacteria liquid processes: step (1) seed of finishing dealing with is soaked in to 10min in bacillus subtilis GB03 bacterium liquid, then bacterium liquid is poured out, the seed of finishing dealing with is sowed.
Embodiment 3: a kind of beneficial rhizosphere bacterium promotes the method for Radix Codonopsis seed germination, and its main feature is that step is:
(1) Radix Codonopsis seed disinfection is processed: the Radix Codonopsis seed of full seed N/D, uniformity, and with after 75% alcohol-pickled 10s, use 5% clorox to soak 12min, the distilled water flushing of use sterilizing 7-8 time, in 4 ℃ of refrigerator overnight;
(2) bacillus subtilis GB03 bacterium solution preparation:
First prepare bacteriolyze meat soup LB liquid nutrient medium: according to the ratio of sodium chloride, tryptone and dusty yeast, be 1:2:1 preparation, solvent is distilled water; Adjust pH to 7.2, high temperature is 125-132 ℃ of sterilizing, after temperature to be cooled is 35 ℃, the LB liquid nutrient medium of every 100mL adds the bacillus subtilis GB03 bacterium liquid of 100 μ L, at rotating speed, be under 180-250r/min, temperature is on the constant-temperature shaking incubator of 30 ℃ after overnight incubation, and mensuration bacterial concentration is OD
600during for 0.8-1.0, take out standby;
(3) planting daughter bacteria liquid processes: step (1) seed of finishing dealing with is soaked in to 20min in bacillus subtilis GB03 bacterium liquid, then bacterium liquid is poured out, the seed of finishing dealing with is sowed.
Experimental example: beneficial rhizosphere bacterium promotes the method for Radix Codonopsis seed germination
1 materials and methods
1.1 material
Radix Codonopsis seed picks up from Minxian County, the Gansu traditional Chinese medicine technological guidance herbal garden of standing in October, 2011, through professor Wang Yinquan of the Gansu college of traditional Chinese medicine, be accredited as the plant Radix Codonopsis Codonopsispilosula of Campanulaceae Codonopsis (Franch.) Nannf., seed is stored in 4 ℃, laboratory refrigerator.Bacillus subtilis GB03 bacterial strain is provided by the Paul W.Par é of Texas, USA Polytechnics professor laboratory, is stored in-80 ℃ of refrigerators.75% alcohol, clorox, sodium chloride are standard reagent.Dusty yeast, tryptone and agar powder are Britain's import reagent.
1.2 experimental technique
1.2.1 seed treatment
Select the Radix Codonopsis seed of full seed N/D, uniformity, in soaking respectively after 10s and 10min in 75% alcohol, 5% clorox for superclean bench, with the distilled water flushing of sterilizing 7-8 time, in 4 ℃ of refrigerator overnight.
1.2.2 the preparation of Bacillus subtillis GB03 bacterium liquid
Preparation LB liquid nutrient medium: according to sodium chloride 5g/L, the ratio preparation of tryptone 10g/L and dusty yeast 5g/L, with standard type pH meter (PB-10, Beijing Sai Duolisi) adjust pH to 7.2, use vertical pressure steam sterilization pan (LDZX-50KBS, Shen, Shanghai peace) high-temperature sterilization after cooling, the LB liquid nutrient medium of every 100mL adds the GB03 bacterium liquid of 100 μ L, at rotating speed, be under 180r/min, temperature is the constant-temperature shaking incubator (THZ-300 of 28 ℃, the permanent science and technology in Shanghai one) after upper overnight incubation, (NanoDrop 1000 to use nucleic acid-protein detector, Germany) mensuration bacterial concentration is OD
600take out standbyly at=0.9 o'clock, bacterium liquid is now with the current.
1.2.3 the experiment of bacterium immersion bubble seed germination
In the centrifuge tube of sterilizing, add cultured GB03 bacterium liquid, then the Radix Codonopsis seed disinfecting is immersed in to (sterile water compares) in bacterium liquid, after timing 5min, 10min and 20min, take out rapidly seed, be laid in the culture dish that sterilizing is covered with two layers of filter paper, before cover plant, on filter paper, splash into equivalent sterile water, till forming moisture film to filter paper surface, the seed of taking-up is layered on filter paper uniformly, 100, every ware, being placed in 25 ℃ of dark conditions cultivates, statistical observation every day germination, adds up germination rate after cultivation 10d, calculates germination vigor.
1.3 data processings and statistical analysis technique
In experiment, so that radicle is prominent, break in the seed coat, show money or valuables one carries unintentionally and be considered as sprouting, observe Radix Codonopsis seed every day and record the seed amount of sprouting the same day, germination rate (GP)=(Gt/T) * 100%, the seed number of Gt for germinateing and ending and all germinate in formula, the sum that T is seed; Germination vigor refers to that Radix Codonopsis germination vigor is defined as the 3d that germinates when the Sino-Japan chitting piece number of seed sprouting process reaches top, and the seed number of germination accounts for the percentage for test sample product seed number.Germination vigor is that (Yang Xiaojie etc. 2003 for the index of judgement field emergence rate; Zhao Lanzhi etc. 2004).At least 6 repetitions of every processing setting, the numerical value in chart is mean value ± SE; Adopt Excel 2003 and SPSS 13.0 to carry out respectively data processing and statistical analysis, by the significance level of difference between each processing of One-way ANOVA check.
2 results and analysis
Germination vigor is the major criterion of weighing seed germination ability, the seed that germination vigor is high, and vitality is strong, higher at the sowing germination rate in land for growing field crops, after planting comes up normal.As seen from Figure 1, sprout the Radix Codonopsis seed germination quantity that the 3rd day bacillus subtilis strain GB03 bacterium liquid of experiment invades after bubble 5min, 10min and 20min more; Invade as can be seen from Figure 2 on the bubble Radix Codonopsis germination seed radicle of 5 minutes root hair obviously more than contrast.As seen from Figure 3, after GB03 bacterium immersion bubble Radix Codonopsis seed 5min, 10min and 20min, contrast all remarkable (P<0.05) and improved Radix Codonopsis potentiality of seed, increase rate is respectively 2.1 times, 1.9 times and 1.5 times, wherein 5min best results, the strongest to the raising ability of germination vigor.As can be seen from Figure 4, after GB03 bacterium immersion bubble Radix Codonopsis seed 5min, 10min and 20min, contrast the germination rate of Radix Codonopsis seed is improved, wherein 5min, 10min soak and improve Radix Codonopsis percentage of seedgermination 8.0% and 2.0%, 20min remarkable (P<0.05) raising germination rate 11.6% (P<0.05).This experimental result shows, bacterium immersion bubble Radix Codonopsis seed 5min facilitation effect the best to seed germination is especially remarkable to the raising of germination vigor.
3 discuss
Germination vigor is more concentrated germination rate of germination initial stage, and it is determining the neat degree of emerging, and germination vigor is high, emerge neat, seedling growth is consistent, otherwise seedling is uneven, when germination rate is identical, the Seed Vitality that germination vigor is high is strong, higher at the sowing germination rate in place, after planting comes up normal, so germination vigor is one of factor of calculating seeding quantity.
The discovery of this result of study, bacillus subtilis bacterium liquid GB03 soaks and has significantly improved Radix Codonopsis potentiality of seed, thereby has improved Radix Codonopsis seed vitality.
In sum, bacillus subtilis strain GB03 bacterium immersion bubble has significantly improved Radix Codonopsis potentiality of seed, more obvious compared with germination rate to the facilitation of germination vigor, thereby Radix Codonopsis seed vitality and emergence rate have been improved, keep more neat germination rate, overcome the upper Radix Codonopsis seed of production little, the problem that germination rate is low, produces thereby reduce the economic losses such as manpower that upper Radix Codonopsis is filled the gaps with seedlings and germination causes repeatedly.The regulation and control of research GB03 to seed germination, more preferably its application in actual production provides theoretical foundation and technological guidance.
4 brief summaries
(1) after bacillus subtilis GB03 bacterium immersion bubble Radix Codonopsis seed 5min, 10min and 20min, contrast all significantly the raising of (P<0.05) germination vigor of Radix Codonopsis seed, increase rate is respectively 211.8%, 192.5% and 145.8%, wherein 5min best results.
(2) after bacillus subtilis GB03 bacterium immersion bubble Radix Codonopsis seed 5min, 10min and 20min, contrast the germination rate of Radix Codonopsis seed is improved, wherein 5min, 10min soak to improve Radix Codonopsis percentage of seedgermination be 8.0% and 2.0%, 20min significantly (P<0.05) to have improved germination rate be 11.6%.
The foregoing is only preferred embodiment of the present invention, not in order to limit the present invention, all any modifications of doing within the spirit and principles in the present invention, be equal to replacement, improvement etc., within all should being included in protection scope of the present invention.
Claims (1)
1. beneficial rhizosphere bacterium promotes a method for Radix Codonopsis seed germination, and its feature comprises the following steps:
(1) Radix Codonopsis seed disinfection is processed: the Radix Codonopsis seed of full seed N/D, uniformity, and with after 75% alcohol-pickled 8-10s, use 5% clorox to soak 10-12min, the distilled water flushing of use sterilizing 7-8 time, in 2-4 ℃ of refrigerator overnight;
(2) bacillus subtilis GB03 bacterium solution preparation: preparation bacteriolyze meat soup LB liquid nutrient medium: be 1:2:1 preparation according to the ratio of sodium chloride, tryptone and dusty yeast, solvent is distilled water; Adjust pH to 7.2, high temperature is 115-132 ℃ of sterilizing, after temperature to be cooled is 20-35 ℃, the LB liquid nutrient medium of every 100mL adds the bacillus subtilis GB03 bacterium liquid of 50-100 μ L, at rotating speed, be under 180-250r/min, temperature is on the constant-temperature shaking incubator of 25-30 ℃ after overnight incubation, and mensuration bacterial concentration is OD
600during for 0.8-1.0, take out standby;
(3) planting daughter bacteria liquid processes: step (1) seed of finishing dealing with is soaked in to 5min-20min in bacillus subtilis GB03 bacterium liquid, then bacterium liquid is poured out, the seed of finishing dealing with is sowed.
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Cited By (5)
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| CN103999595A (en) * | 2014-06-05 | 2014-08-27 | 兰州大学 | Method for improving salt tolerance of codonopsis pilosula |
| CN104255110A (en) * | 2014-09-04 | 2015-01-07 | 兰州大学 | Method for promoting germination of alfalfa seeds |
| CN104396736A (en) * | 2014-09-23 | 2015-03-11 | 兰州大学 | Method for improving germination rate of Puccinellia tenuiflora seed |
| CN107549636A (en) * | 2017-09-13 | 2018-01-09 | 安徽鑫泉米业有限公司 | A kind of method that beneficial bacterium fermentating metabolism product improves brown rice germination rate and sprouted unpolished rice quality |
| CN115380663A (en) * | 2022-07-25 | 2022-11-25 | 山西农业大学经济作物研究所 | Seed sterilization method for codonopsis pilosula regeneration system |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN115380663A (en) * | 2022-07-25 | 2022-11-25 | 山西农业大学经济作物研究所 | Seed sterilization method for codonopsis pilosula regeneration system |
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