CN103828721B - Bacillus licheniformis is promoting the application in blueberry tissue culture seedling rooting or growth - Google Patents
Bacillus licheniformis is promoting the application in blueberry tissue culture seedling rooting or growth Download PDFInfo
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- CN103828721B CN103828721B CN201410105267.7A CN201410105267A CN103828721B CN 103828721 B CN103828721 B CN 103828721B CN 201410105267 A CN201410105267 A CN 201410105267A CN 103828721 B CN103828721 B CN 103828721B
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Abstract
The invention discloses bacillus licheniformis and promote the application in blueberry tissue culture seedling rooting or growth, described application comprises: direct is raw material with Zheng Chang Sheng, after bacterium activation, be seeded on blueberry Regenerated plant with the form of Bacillus licheniformis liquid, eliminate the step of a large amount of separation, purifying, qualification bacterial strain, also do not add any exogenous hormone, with low cost, simple to operate, safety non-toxic, successful; Postvaccinal blueberry tissue culture seedling rooting rate significantly improves, and the index such as plant height, leaf area also improves, and after fungal contamination, survival rate also increases substantially, and significantly improves the quality of rooting of blueberry tissue culture seedling.
Description
Technical field
The present invention relates to the application of bacillus licheniformis, particularly relating to bacillus licheniformis promoting the application in plant tissue culture seedling rooting or growth, belonging to the application of bacillus licheniformis.
Background technology
Blueberry (SemenTrigonellae) has softening human vas, strengthens the health care effectiveness such as immunity function, is described as " king of fruit ", and also food and agricultural organization of united state is classified as one of large healthy food of the mankind five, and the market price is expensive.For now, blueberry output can not meet domestic demand far away, and the various converted products of blueberry are well received in international market.But because its material source is limited, reproduction coefficient is low, cannot accomplish scale production, tissue cultures is the important channel addressed this problem.But conventional method expands numerous popularization slowly, blueberry rooting of vitro seedling is difficult, rooting rate is low, the root of hair cycle is long, constrains the industrialization nursery process of improved seeds.
Summary of the invention
In order to overcome above-mentioned prior art Problems existing, the object of this invention is to provide bacillus licheniformis and promoting the application in blueberry tissue culture seedling rooting or growth.
Described application inoculates bacillus licheniformis on blueberry Regenerated plant, carries out sugar free tissue culture.
Particularly, described bacillus licheniformis take Zheng Chang Sheng as raw material, after bacterium activation process, is seeded on blueberry Regenerated plant with the form of Bacillus licheniformis liquid.
Especially, described bacterium activation process comprises following steps:
By bacillus licheniformis dry powder, after being inoculated in liquid nutrient medium, carry out first time vibration, duration of oscillation is 2-3 hour, and design temperature is 30 DEG C, and rotating speed is 180 revs/min;
Be inoculated on solid culture medium by the bacterium liquid completing first time vibration, cultivate 48 hours, get single bacterium colony in liquid nutrient medium in the constant incubator of 28 DEG C, carry out second time vibration, duration of oscillation is 8 hours, and design temperature is 30 DEG C, and rotating speed is 180 revs/min;
By complete second time vibration bacterium liquid carry out centrifugal treating, discard supernatant, at the temperature of-20 DEG C, with concentration be 10% glycerine frozen.
Wherein, described liquid nutrient medium and solid culture medium are LB medium.
The formula of LB liquid nutrient medium is: yeast extract 5g/L, tryptone 10g/L, sodium chloride 10g/L, pH6.8-7.2.
The formula of LB solid culture medium is: yeast extract 5g/L, tryptone 10gg/L, sodium chloride 10gg/L, agar 17-20gg/L, pH6.8-7.2.
Particularly, the concentration of described Bacillus licheniformis liquid is 100000cfu/ml.
Especially, describedly on blueberry Regenerated plant, inoculate bacillus licheniformis comprise the blueberry Regenerated plant of well cutting is soaked in described Bacillus licheniformis liquid, immersion treatment 5min.
Particularly, described sugar free tissue culture comprises: inserted in blueberry sugar free tissue culture base by the blueberry Regenerated plant of inoculation bacillus licheniformis, carry out tissue cultures.
Especially, the formula of described blueberry sugar free tissue culture base is: potassium nitrate 1900mg/L, ammonium nitrate 1650mg/L, monobasic potassium phosphate 170mg/L, magnesium sulfate 370mg/L, calcium chloride 440mg/L, potassium iodide 0.83mg/L, boric acid 6.2mg/L, manganese sulphate 22.3mg/L, zinc sulphate 8.6mg/L, nickel acid sodium 0.25mg/L, copper sulphate 0.25mg/L, cobalt chloride 0.025mg/L, disodium ethylene diamine tetraacetate 37.25mg/L, ferrous sulfate 27.85mg/L, thiamine hydrochloride 1mg/L, agar 6g/L, pH=5.8.
The present invention compared with prior art, has following beneficial outcomes:
Bacillus licheniformis is applied to the link of taking root of the blueberry promoting sugar free tissue culture by the present invention first, and the rooting rate of blueberry tissue culture seedling is significantly improved;
Bacillus licheniformis used in the present invention is directly raw material with Zheng Chang Sheng, after bacterium activation, be seeded on blueberry Regenerated plant with the form of Bacillus licheniformis liquid, eliminate the step of a large amount of separation, purifying, qualification bacterial strain, any exogenous hormone is not added yet, with low cost, simple to operate, safety non-toxic, successful.
According to the blueberry tissue culture seedling of technical solutions according to the invention process, other indexs such as plant height, leaf area also improve, and after fungal contamination, survival rate also increases substantially, and significantly improve the quality of rooting of blueberry tissue culture seedling.
Embodiment
Further describe the beneficial effect of technical scheme of the present invention below by embodiment, these embodiments are only exemplary, do not form any restriction to scope of the present invention.It will be understood by those skilled in the art that and do not departing under formula thinking of the present invention, purposes scope and can modify to the details of technical solution of the present invention and form or replace, but these amendments and replacement all fall within the scope of protection of the present invention.
The test method used in following embodiment, if no special instructions, is conventional method.
Percentage composition in following embodiment, if no special instructions, is percentage composition.
The material used in following embodiment, reagent etc., if no special instructions, all can obtain from commercial channels.
The preparation of Preparative Example 1 Bacillus licheniformis liquid
1) Zheng Chang Sheng (i.e. bacillus licheniformis capsule is opened, manufacturer: Northeast Pharmaceutical Group Shenyang First Pharmaceutical Co., Ltd.) capsule shell, bacillus licheniformis dry powder is wherein dipped with oese, be inoculated in the conical flask that LB liquid nutrient medium is housed, put into shaking table and carry out first time vibration, duration of oscillation is 2-3 hour, and design temperature is 30 DEG C, and rotating speed is 180 revs/min.
Wherein, the formula of described LB liquid nutrient medium is: yeast extract 5g/L, tryptone 10g/L, sodium chloride 10g/L, pH6.8-7.2.
2) after having vibrated for the first time, bacterium liquid is dipped with oese, even streak inoculation is on LB solid culture medium, cultivate 48 hours in the constant incubator of 28 DEG C, get single bacterium colony in the conical flask that LB liquid nutrient medium is housed, put into shaking table and carry out second time vibration, duration of oscillation is 8 hours, design temperature is 30 DEG C, and rotating speed is 180 revs/min.
Wherein, the formula of described LB solid culture medium is: yeast extract 5g/L, tryptone 10gg/L, sodium chloride 10gg/L, agar 17-20gg/L, pH6.8-7.2.
3), after second time has been vibrated, centrifugal treating has been carried out to bacterium liquid, has discarded supernatant, at the temperature of-20 DEG C, be the frozen residue paste of glycerine of 10% by concentration, make Bacillus licheniformis liquid, for subsequent use.
Embodiment 1 bacillus licheniformis is promoting the application in blueberry tissue culture seedling rooting or growth
1. inoculate:
In the culture dish of diameter 35mm, the concentration that the Bacillus licheniformis liquid aseptic deionized water made by Preparative Example 1 is diluted to wherein bacillus licheniformis is 100000cfu/ml;
The blueberry Regenerated plant (2-3cm) of well cutting is soaked in above-mentioned culture dish, immersion treatment 5min;
2 tissue cultures:
Inserted by blueberry Regenerated plant after immersion treatment in blueberry sugar free tissue culture base, carry out tissue cultures, incubation time is 8 weeks, obtains blueberry tissue culture seedling.
Wherein, the formula of described blueberry sugar free tissue culture base is: potassium nitrate 1900mg/L, ammonium nitrate 1650mg/L, monobasic potassium phosphate 170mg/L, magnesium sulfate 370mg/L, calcium chloride 440mg/L, potassium iodide 0.83mg/L, boric acid 6.2mg/L, manganese sulphate 22.3mg/L, zinc sulphate 8.6mg/L, nickel acid sodium 0.25mg/L, copper sulphate 0.25mg/L, cobalt chloride 0.025mg/L, disodium ethylene diamine tetraacetate 37.25mg/L, ferrous sulfate 27.85mg/L, thiamine hydrochloride 1mg/L, agar 6g/L, pH=5.8.
Comparative examples 1
Get the blueberry Regenerated plant (2-3cm) consistent with upgrowth situation in embodiment 1 and be soaked in 5 minutes (with the Bacillus licheniformis liquid same volume of diluting in embodiment 1) in aseptic deionized water, then be inoculated in blueberry sugar free tissue culture base, carry out tissue cultures, incubation time is 8 weeks, obtains blueberry tissue culture seedling.
Test example 1 grows and rooting inhibitors measures
1.1 assay methods:
Each 30 of blueberry tissue culture seedling described in Example 1 and reference examples 1, from cultivating beginning in the 3rd week, observes its situation calculate rooting rate of taking root; After cultivating eight weeks, start to measure plant height, leaf area, root length and radical four growth indexes, and based on above index, evaluate the quality of rooting of blueberry tissue culture seedling.
Test in triplicate, take the mean, and calculate standard error.
Number/blueberry tissue culture seedling sum × 100% of the blueberry tissue culture seedling of rooting rate=taken root;
Quality of rooting Q=rooting rate (the 8th week) × 50%+ radical × 25%+ root long × 25%.
1.2 measurement results:
1.2.1 rooting rate
Observe the rooting rate of blueberry tissue culture seedling described in embodiment 1 and reference examples 1 and calculate, result is as shown in table 1.
The rooting rate of the different blueberry tissue culture seedling of table 1
As shown in Table 1, the blueberry tissue culture seedling that embodiment 1 is turned out, in whole incubation, rooting rate is all higher than the blueberry tissue culture seedling that reference examples 1 is cultivated.
1.2.2 growth indexes
To the plant height of blueberry tissue culture seedling described in embodiment 1 and reference examples 1, leaf area, root, long and radical four growth indexes measure, and result is as shown in table 2.
The growth indexes of the different blueberry tissue culture seedling of table 2
As shown in Table 2, the blueberry tissue culture seedling that embodiment 1 is turned out, every growth indexes is all obviously better than the blueberry Regenerated plant that control group 1 is turned out, compared with the blueberry tissue culture seedling of turning out with reference examples 1, its plant height, leaf area, root are long, radical adds 175%, 135%, 173%, 161% respectively.What bacillus licheniformis was described uses the growing state that obviously can improve blueberry Regenerated plant.
Meanwhile, dig up the roots long outside, the standard error of other three indexs of the blueberry tissue culture seedling that embodiment 1 is turned out all is less than the blueberry tissue culture seedling that reference examples 1 is turned out, and illustrates that the growth uniformity of the blueberry tissue culture seedling using Bacillus licheniformis liquid is better.
1.2.3 quality of rooting
Based on These parameters, evaluate the quality of rooting of blueberry tissue culture seedling described in embodiment 1 and reference examples 1, result is as shown in table 3.
The quality of rooting of the different blueberry tissue culture seedling of table 3
| Embodiment 1 | Reference examples 1 | |
| Quality of rooting | 3.301 | 1.44 |
| Standard error | 0.0551 | 0.0616 |
As shown in Table 3, the quality of rooting of the blueberry tissue culture seedling that embodiment 1 is turned out significantly is better than the quality of rooting of the blueberry tissue culture seedling that reference examples 1 is turned out, and the former is 2.3 times of the latter; And grow uniformity aspect, be also the embodiment 1 blueberry tissue culture seedling of turning out slightly well.
In sum, Bacillus licheniformis liquid is taken root for blueberry sugar-free plantlet in vitro obvious facilitation, and can significantly improve plantlet in vitro quality.
Claims (4)
1. bacillus licheniformis is promoting the application in blueberry tissue culture seedling rooting or growth, it is characterized in that: carry out tissue cultures by after blueberry Regenerated plant inoculation bacillus licheniformis;
Wherein, described by blueberry Regenerated plant inoculation bacillus licheniformis also comprise: by bacillus licheniformis kind after bacterium activation process, be seeded on blueberry Regenerated plant with the form of Bacillus licheniformis liquid;
Wherein, carrying out tissue cultures described in is sugar free tissue culture;
Wherein, described sugar-free culture-medium is potassium nitrate 1900mg/L, ammonium nitrate 1650mg/L, monobasic potassium phosphate 170mg/L, magnesium sulfate 370mg/L, calcium chloride 440mg/L, potassium iodide 0.83mg/L, boric acid 6.2mg/L, manganese sulphate 22.3mg/L, zinc sulphate 8.6mg/L, nickel acid sodium 0.25mg/L, copper sulphate 0.25mg/L, cobalt chloride 0.025mg/L, disodium ethylene diamine tetraacetate 37.25mg/L, ferrous sulfate 27.85mg/L, thiamine hydrochloride 1mg/L, agar 6g/L, pH=5.8.
2. application according to claim 1, is characterized in that, described bacterium activation process comprises following steps:
By bacillus licheniformis dry powder, after being inoculated in liquid nutrient medium, carry out first time vibration, duration of oscillation is 2-3 hour, and temperature is 30 DEG C, and rotating speed is 180 revs/min;
Be inoculated on solid culture medium by the bacterium liquid completing first time vibration, cultivate 48 hours, get single bacterium colony in liquid nutrient medium in the constant incubator of 28 DEG C, carry out second time vibration, duration of oscillation is 8 hours, and design temperature is 30 DEG C, and rotating speed is 180 revs/min;
By complete second time vibration bacterium liquid carry out centrifugal treating, discard supernatant, at the temperature of-20 DEG C, with concentration be 10% glycerine frozen.
3. application according to claim 2, is characterized in that, the concentration of described Bacillus licheniformis liquid is 100000cfu/ml.
4. application according to claim 2, is characterized in that: described inoculation comprises and is soaked in Bacillus licheniformis liquid by the blueberry Regenerated plant of well cutting, immersion treatment 5min.
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