CN109932229A - A kind of collection method of Rhizosphere of Crops secretion - Google Patents
A kind of collection method of Rhizosphere of Crops secretion Download PDFInfo
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- CN109932229A CN109932229A CN201910222390.XA CN201910222390A CN109932229A CN 109932229 A CN109932229 A CN 109932229A CN 201910222390 A CN201910222390 A CN 201910222390A CN 109932229 A CN109932229 A CN 109932229A
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Abstract
The present invention relates to field of biotechnology more particularly to a kind of collection methods of Rhizosphere of Crops secretion.Collection method uses following steps: placing two layers of filter paper in culture dish and is drenched with sterile distilled water；Pretreated corn seed is placed in presprouting in culture dish；After germination, the consistent Western corn of growing way is moved in the water planting container equipped with culture solution and is cultivated, guarantees that the intact nothing in root is impaired；It is long nutrient solution to be replaced with into sterile distilled water, 6h, which is moved back, collects root rhizosphere secreta into superclean bench after illumination period starts 2h to four leaf stage.The root rhizosphere secreta that this experimental method is collected is extremely successful, it is not necessary that corn seedling is removed culture apparatus in experiment, effectively prevents the damage to maize root system, and then ensure that the accuracy of experimental result；Strict sterilization, sterilizing change replacement nutrient solution frequently, avoid seedling microbiological contamination and microbial contamination etc..More previous culture collection method has the advantages that efficient, accurate.
Description
Technical field
The present invention relates to field of biotechnology more particularly to a kind of collection methods of Rhizosphere of Crops secretion.
Background technique
Since self-discovery Rhizospheric microorganisms' quantity is more, type is few and the growth and development of crop has close relationship, to it
Research has obtained more and more attention.Further research to rhizosphere microorganism, more than illustrate rhizosphere microorganism quantity,
Type and its mechanism of action, it is often more important that it will for arrange crop rotation, make etc. tillage control measures and it is economical with fertilizer provide science according to
According to there is important meaning in agricultural production.
Many scholars, which have studied plant root secretion and rhizosphere microorganism, in recent years close relationship, it has been found that plants
Object secretes many organic matters and the inorganic matters such as carbon dioxide, sugar, organic acid, alcohol, itrogenous organic substance matter from root in growth period,
These substances are the good nutriment of rhizosphere microorganism, but the secretion requirement to root secretion, mechanism and its plant from soil
Effect during earth absorption nutrient still lacks in-depth study.It will be evident from crop to the research of root secretion to absorb
The mechanism of nutritive process and how to effectively utilize the nutriment in soil.
The culture of Rhizosphere of Crops secretion at present mostly uses bulky plastic box device, and cultivating system is larger, the plant of culture
Strain number amount is more, is continuously ventilated using venting in timing manner device, and the replacement number of nutrient solution is once a week.Existing method is easy
Cause plant rhizosphere to pollute, leads to the root rhizosphere secreta ingredient inaccuracy collected.
Summary of the invention
Based on defect of the existing technology, one of research purpose of the invention is to provide a kind of Rhizosphere of Crops secretion
Collection method, the method provide one kind and do not disturb root growth, do not destroy rhizospheric environment, monitor rhizosphere pathogen infection at any time
The method of root system object variation afterwards, it is therefore intended that overcome in existing method root system it is perishable, vulnerable to microbial contamination, sampling easily injury
Root system is unable to the drawbacks such as continuous sampling.
A kind of collection method of Rhizosphere of Crops secretion, using following steps:
(1) places two layers of filter paper in culture dish and is drenched with sterile distilled water；
(2) pretreated corn seed is placed in presprouting in culture dish by；
(3) is after germination, and the consistent Western corn of growing way is moved to the water planting container equipped with culture solution, and (floor space is
9.6cm2, a height of 12cm) in culture, guarantee that the intact nothing in root is impaired；
(4) nutrient solution is replaced with sterile distilled water, 6h is moved back to ultra-clean work after illumination period starts 2h to four leaf stage by long
Root rhizosphere secreta is collected in platform.
Preferably, step (1) the culture dish floor space is 9.0-10.0cm2, a height of 11-13cm；The water planting culture
Ware volume system is 100ml.
Preferably, the preprocess method of pretreated corn seed described in step (2) are as follows: be placed in corn seed
3min is shaken in 96% ethyl alcohol, 30 min are shaken in 3% liquor natrii hypochloritis, and sterile distillation twice is rinsed in sterile distilled water
4 h are impregnated in water at 25 DEG C.
Preferably, presprouting described in step (2) is the dark processing to germination at 28 DEG C.
Preferably, the actual conditions cultivated in hydroponic device described in step (4) are as follows: one time of nutrition liquid is every other day changed,
8h is cultivated at 20 DEG C in the dark state, then cultivates 16 h at 25 DEG C under illumination conditions, and dark state and illumination conditions are handed over
For culture.
Preferably, the method for root rhizosphere secreta is collected described in step (4) are as follows: draw the liquid in culture dish with syringe
Body crosses 0.45um film, dispenses into batch cultur ware, -80 DEG C of storages, freeze-drying to pulverulence.
Preferably, the nutrient solution is Hoagland ' s culture solution.
Beneficial effect
The present invention by principal component analysis figure and difference metabolin hierarchical clustering result it can be found that, the root that this experimental method is collected
Border secretion is extremely successful, uses cultivating system for the small system of 100mL, and two plants of seedling are cultivated in each device, avoid intersection
Pollution；It is not necessary that corn seedling is removed culture apparatus in experiment, the damage to maize root system is effectively prevented, and then ensure that reality
Test the accuracy of result；Strict sterilization, sterilizing change replacement nutrient solution frequently, avoid seedling microbiological contamination and microbial contamination etc..Relatively first
Preceding culture collection method has the advantages that efficient, accurate.
Detailed description of the invention
Fig. 1 is the sample PCA shot chart that 1 method of the embodiment of the present invention is collected；
Fig. 2 is the sample PCA shot chart that conventional method is collected in comparative example 1；
Fig. 3 is the difference metabolin that collection method of the present invention detects；
Fig. 4 is the difference metabolin that conventional acquisition method detects；
Fig. 5 is the tri-leaf period B73 seedling of the method for the present invention culture.
Specific embodiment
Technical solution of the present invention is further explained in following embodiment, according to above description and these implementations
Example, those skilled in the art can determine essential characteristic of the invention, and without departing from spirit and scope of the invention the case where
Under, various changes and modifications can be made to the present invention, so that it is applicable in various uses and condition.
Embodiment 1
The collection method of Rhizosphere of Crops secretion:
1. preparing 1 × Hoagland ' s culture solution, hydroponic device impregnates for 24 hours in 7% liquor natrii hypochloritis；In culture dish
It places two layers of filter paper and is drenched with sterile distilled water；
2. high-temperature sterilization Hoagland ' s culture solution, glass culture dish (including filter paper)；The culture dish floor space is
9.6cm2, a height of 12cm；The water planting culture dish volume system is 100ml；2 plants of corn seedlings are cultivated in each culture dish.
Have sprouting condition, 40, uniform B73 seed 3. selecting, is placed in 96% ethyl alcohol and shakes 3min, 3%
30 min are shaken in liquor natrii hypochloritis, are rinsed twice in sterile distilled water, are impregnated 4 h in sterile distilled water at 25 DEG C；
4. B73 seed is placed in presprouting in culture dish, and 28 DEG C, dark processing；
5., by the consistent Western corn of growing way (16 plants of B73), moving to cultivated equipped with Hoagland ' s respectively after germination
Culture in the hydroponic device (every two plants co-culture) of liquid, guarantees that the intact nothing in root is impaired, every other day replacement one time of nutrition liquid, 20
8 h are dark at DEG C, 16 h illumination at 25 DEG C；
6. long replace with the sterile steaming of 100mL for 100mL Hoagland ' s nutrient solution after illumination period starts 2h to four leaf stage
Distilled water, 6h, which is moved back, collects root rhizosphere secreta into superclean bench；
7. using Dispoable medical syringe, the liquid in culture apparatus is drawn, crosses 0.45um film, packing to batch cultur ware
In, -80 DEG C of storages, freeze-drying to pulverulence.
8. LC-MS is analyzed.
Comparative example 1
1. nutrient solution is Hoagland nutrient solution, nutrition device uses capacity for the round plastic bucket of 10 L, with iron wire frame and rubber
Muscle is made into supporting network.
2. H2O2The B73 seed sterilized is sowed at saturation CaSO4In the quartz sand that solution impregnation is crossed, 28 DEG C of conditions
Under be protected from light germination, irradiation culture when cotyledon exposes, when root long is to 3cm-5cm, careful take out grows uniform seedling, spend from
After sub- water gently washes away quartz sand, it is placed in 1/2Hoagland nutrient solution and is colonized.
3. 1 plant of every hole, density is 15 plants/basin, and the mesh for not being colonized seedling is covered with dark colored plastic film.Plant culture period
Between periodically ventilate.At the end of 1st week, nutrient solution is changed to complete nutrition liquid, is hereafter replaced nutrient solution 1 time weekly, guarantees seedling
Normal growth, 8 h are dark at 20 DEG C, 16 h illumination at 25 DEG C.
4. by the CaCl of the consistent four leaf stage seedling replanting of growing way to 6 L, 0.5 mmol/L2In solution, in room temperature and light
4 h (9: 00-13: 00) is cultivated under the conditions of line is good, with 500 mL CH2Cl2Solution extracts root washing lotion 2 times, by extracting solution mistake
0.45 μm of film, freeze-drying to pulverulence.
5. LC-MS is analyzed.
2 experimental result of embodiment
The B73 root rhizosphere secreta collected in the embodiment of the present invention 1 and comparative example 1, LC-MS interpretation of result discovery:
By Fig. 1 and Fig. 2 it can be found that there is one group of data more to deviate in the data of the conventional method in comparative example 1, and it is of the invention
The data degree of fitting that method obtains is very high, and the model is more reliable.
The metabolin of VIP>1 and univariate statistics analysis value<0.05 P is can be found that by Fig. 3 and Fig. 4, as
Metabolin with significant difference；And VIP>1 and value<0.1 item 0.05<P conduct difference metabolin.
Claims (7)
1. a kind of collection method of Rhizosphere of Crops secretion, which is characterized in that use following steps:
(1) places two layers of filter paper in culture dish and is drenched with sterile distilled water；
(2) pretreated corn seed is placed in presprouting in culture dish by；
(3) is after germination, and the consistent Western corn of growing way is moved to the water planting container equipped with culture solution, and (floor space is
9.6cm2, a height of 12cm) in culture, guarantee that the intact nothing in root is impaired；
(4) nutrient solution is replaced with sterile distilled water, 6h is moved back to ultra-clean work after illumination period starts 2h to four leaf stage by long
Root rhizosphere secreta is collected in platform.
2. collection method according to claim 1, which is characterized in that step (1) the culture dish floor space is 9.0-
10.0cm2, a height of 11-13cm；The water planting culture dish volume system is 100ml；2 plants of corn childrens are cultivated in each culture dish
Seedling.
3. collection method according to claim 1, which is characterized in that pretreated corn seed described in step (2)
Preprocess method are as follows: corn seed is placed in 96% ethyl alcohol and shakes 3min, 30 min are shaken in 3% liquor natrii hypochloritis,
It is rinsed in sterile distilled water twice, impregnates 4 h in sterile distilled water at 25 DEG C.
4. collection method according to claim 1, which is characterized in that presprouting described in step (2) is the dark at 28 DEG C
It handles to germination.
5. collection method according to claim 1, which is characterized in that the tool cultivated in hydroponic device described in step (4)
Concrete conditions in the establishment of a specific crime are as follows: every other day change one time of nutrition liquid, 8h is cultivated at 20 DEG C in the dark state, then under illumination conditions at 25 DEG C
Cultivate 16 h, dark state and illumination conditions alternate culture.
6. collection method according to claim 1, which is characterized in that collect the side of root rhizosphere secreta described in step (4)
Method are as follows: draw the liquid in culture dish with syringe, cross 0.45um film, dispense into batch cultur ware, -80 DEG C of storages are cold
It is lyophilized dry to pulverulence.
7. collection method described in -6 according to claim 1, which is characterized in that the nutrient solution is Hoagland ' s culture solution.
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Citations (5)
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|---|---|---|---|---|
| US4061488A (en) * | 1972-11-13 | 1977-12-06 | Hershey Foods Corporation | Plant treating mixtures and methods utilizing spores of Bacillus uniflagellatus |
| CN103141368A (en) * | 2013-03-11 | 2013-06-12 | 天津师范大学 | Transparent device and method for directly observing relation between plant root growth and microorganism |
| CN103759978A (en) * | 2014-02-10 | 2014-04-30 | 金陵科技学院 | Multi-combined device for sterile collection of plant root exudates and application thereof |
| CN104351047A (en) * | 2014-08-28 | 2015-02-18 | 中国农业科学院农业资源与农业区划研究所 | Preparation method of soybean root exudate for efficiently promoting competitive nodulation of rhizobium japonicum |
| CN104858223A (en) * | 2015-05-06 | 2015-08-26 | 同济大学 | Soil metal oxide nanometer material polluted plant repair method |
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2019
- 2019-03-22 CN CN201910222390.XA patent/CN109932229A/en active Pending
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US4061488A (en) * | 1972-11-13 | 1977-12-06 | Hershey Foods Corporation | Plant treating mixtures and methods utilizing spores of Bacillus uniflagellatus |
| CN103141368A (en) * | 2013-03-11 | 2013-06-12 | 天津师范大学 | Transparent device and method for directly observing relation between plant root growth and microorganism |
| CN103759978A (en) * | 2014-02-10 | 2014-04-30 | 金陵科技学院 | Multi-combined device for sterile collection of plant root exudates and application thereof |
| CN104351047A (en) * | 2014-08-28 | 2015-02-18 | 中国农业科学院农业资源与农业区划研究所 | Preparation method of soybean root exudate for efficiently promoting competitive nodulation of rhizobium japonicum |
| CN104858223A (en) * | 2015-05-06 | 2015-08-26 | 同济大学 | Soil metal oxide nanometer material polluted plant repair method |
Non-Patent Citations (3)
| Title |
|---|
| K.J.YOSHITOMI等: "Corn (Zea mays L.) root exudates and their impact on 14C-pyrene mineralization", 《SOIL BIOLOGY & BIOCHEMISTRY》 * |
| 朱建堂: "玉米Sep15-like调控内质网应激响应逆境胁迫的研究", 《中国优秀博士学位论文全文数据库农业科技辑》 * |
| 柴强等: "玉米根系分泌物的分离鉴定及典型分泌物的化感效应", 《甘肃农业大学学报》 * |
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