CN114698512B - Application of nano cerium oxide in promoting plant lateral root occurrence - Google Patents
Application of nano cerium oxide in promoting plant lateral root occurrence Download PDFInfo
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01G—HORTICULTURE; CULTIVATION OF VEGETABLES, FLOWERS, RICE, FRUIT, VINES, HOPS OR SEAWEED; FORESTRY; WATERING
- A01G22/00—Cultivation of specific crops or plants not otherwise provided for
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01C—PLANTING; SOWING; FERTILISING
- A01C1/00—Apparatus, or methods of use thereof, for testing or treating seed, roots, or the like, prior to sowing or planting
- A01C1/08—Immunising seed
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01C—PLANTING; SOWING; FERTILISING
- A01C21/00—Methods of fertilising, sowing or planting
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- C—CHEMISTRY; METALLURGY
- C05—FERTILISERS; MANUFACTURE THEREOF
- C05D—INORGANIC FERTILISERS NOT COVERED BY SUBCLASSES C05B, C05C; FERTILISERS PRODUCING CARBON DIOXIDE
- C05D9/00—Other inorganic fertilisers
- C05D9/02—Other inorganic fertilisers containing trace elements
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- C—CHEMISTRY; METALLURGY
- C05—FERTILISERS; MANUFACTURE THEREOF
- C05G—MIXTURES OF FERTILISERS COVERED INDIVIDUALLY BY DIFFERENT SUBCLASSES OF CLASS C05; MIXTURES OF ONE OR MORE FERTILISERS WITH MATERIALS NOT HAVING A SPECIFIC FERTILISING ACTIVITY, e.g. PESTICIDES, SOIL-CONDITIONERS, WETTING AGENTS; FERTILISERS CHARACTERISED BY THEIR FORM
- C05G3/00—Mixtures of one or more fertilisers with additives not having a specially fertilising activity
Abstract
The invention provides an application of nano cerium oxide in promoting plant lateral root generation, belonging to the technical field of fertilizers. The nano cerium oxide is negatively charged nano cerium oxide. The nano cerium oxide provided by the invention can effectively promote the occurrence of plant lateral roots, thereby promoting the growth and development of plants.
Description
Technical Field
The invention belongs to the technical field of fertilizers, and particularly relates to application of nano cerium oxide in promoting plant lateral root generation.
Background
Under the conditions of continuously increasing population and gradually decreasing cultivated land, the requirements on crop yield and quality are higher and higher. The root system is taken as one of the main organs of the plant, and the good and strong root system is the basis for the rapid growth and the high yield and the high harvest of crops. At present, the modes for promoting root growth mainly comprise: (1) applying a rooting agent and an auxin analogue for external use; (2) reinforcing cultivation management measures; (3) The genetic breeding means breeds developed root varieties or genetically engineered improved crops.
However, the measures have the problems of low absorption and utilization rate, resource waste, unfriendly environment, long time, difficult improvement and the like. In recent years, the application of nanomaterials in crop production has received increasing attention. The nano pesticide has the function in the field of plant protection through the development of the nano pesticide, and the application of the nano fertilizer thoroughly changes the agricultural nutrition form. Various nanomaterials have been reported to regulate plant growth, improve nutritional quality and growth rate, but the application of nanomaterials in plant roots and the effect on side root development are poorly understood. It is important to explore a proper nano material and application mode to promote root growth.
Disclosure of Invention
The invention provides an application of nano cerium oxide in promoting plant lateral root generation, which can effectively promote plant lateral root generation and further promote plant growth.
The invention provides application of nano cerium oxide in root system applied fertilizer for promoting plant lateral root generation, wherein the nano cerium oxide is negatively charged nano cerium oxide.
Preferably, the particle size of the negatively charged nano cerium oxide is 1-10 nm.
Preferably, the preparation method of the negatively-charged nano cerium oxide comprises the following steps:
mixing an aqueous solution of cerium nitrate and an aqueous solution of polyacrylic acid to obtain a mixed solution;
and mixing the mixed solution with an ammonium hydroxide solution with the mass concentration of 30% for reaction to obtain the negatively charged nano cerium oxide.
Preferably, the mass ratio of the cerium nitrate to the polyacrylic acid is 1-2:5-6.
Preferably, the mass volume ratio of the cerium nitrate to the ammonium hydroxide solution with the mass concentration of 30% is 1-2 g:12-18 mL.
Preferably, the reaction time is 20-28 h and the temperature is 20-30 ℃.
Preferably, the application concentration of the negatively-charged nano cerium oxide is 0.05-0.1 mM.
Compared with the prior art, the invention has the advantages and positive effects that:
the root system application fertilizer for promoting the plant lateral root generation provided by the invention has the functional components of nano cerium oxide, wherein the nano cerium oxide can change H in the root system 2 O 2 Content and O 2 - Distribution of (c) and thus O 2 - Accumulating in the early development stage (stages 1-4) of the lateral root primordium of the mature region and the appearance stage of the lateral root primordium, and promoting the occurrence of lateral root.
Drawings
FIG. 1PNC, ceO in example 1 2 TEM images of NPs;
FIG. 2 is a graph showing the characteristic absorption peaks of PNC, dil-PNC, ANC measured by ultraviolet-visible spectrophotometry in example 1;
FIG. 3 shows PNC, ANC and CeO in example 1 2 Particle size of NPs;
FIG. 4 shows PNC, ANC and CeO in example 1 2 The potential of NPs;
FIG. 5 is a graph showing the results of treating root systems of Arabidopsis seedlings with various cerium oxide nanoparticles in example 2; wherein, figure A is a 7d phenotype chart of the root system of Arabidopsis seedlings treated by different cerium oxide nano particles; b, treating the number of 7d lateral roots of the root system of the Arabidopsis seedling by using different cerium oxide nano particles; c, treating the root systems of the arabidopsis seedlings with different cerium oxide nano particles for 7d dry fresh weight;
FIG. 6 is a graph showing H in root system of PNC treated Arabidopsis thaliana 7d in example 2 2 O 2 The content is as follows; wherein A is DCF staining to quantify H in root system 2 O 2 The method comprises the steps of carrying out a first treatment on the surface of the B, counting DCF fluorescence values; c H in main root and lateral root of Arabidopsis thaliana 2 O 2 The content is as follows;
FIG. 7 is a graph showing the presence of O in root system of Arabidopsis thaliana 7d after PNC treatment in example 2 2 - The content is as follows; wherein A, DHE staining quantifies H in root systems 2 O 2 The method comprises the steps of carrying out a first treatment on the surface of the B, counting DHE fluorescence values; c, O in main root and lateral root of Arabidopsis thaliana 2 - The content is as follows;
FIG. 8 is the effect of PNC administration at various concentrations on Arabidopsis thaliana lateral root development in example 2; wherein, A, the root length and lateral root density of the Arabidopsis thaliana after 7d treatment by PNC; PNC treated Arabidopsis 7d later phenotype map;
FIG. 9 is a PNC localization map of DiI-PNC in root system of Arabidopsis thaliana in example 2;
FIG. 10 is a phenotype plot and lateral root scan plot of rice with PNC applied at different concentrations in example 3;
FIG. 11 is a phenotype of canola applied at different concentrations of PNC in FIG. 4, scanned on lateral roots.
Detailed Description
The following description of the technical solutions in the embodiments of the present invention will be clear and complete, and it is obvious that the described embodiments are only some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
The invention provides application of nano cerium oxide in promoting plant lateral root generation, wherein the nano cerium oxide is negatively charged nano cerium oxide.
In the invention, the negatively charged nano cerium oxide enters the primary root and the lateral root primordial positions to remove H in the root system 2 O 2 Content and promote O 2 - Accumulation in the lateral root primordia of the mature region promotes the development of Arabidopsis lateral roots.
In the invention, the nano cerium oxide can be independently applied to crops to promote the generation of plant lateral roots, and can also be mixed with other components to prepare fertilizer, nutrient solution and the like to promote the generation of plant lateral roots.
In the present invention, the particle size of the negatively charged nano cerium oxide is 1 to 10nm.
In the present invention, the preparation method of the negatively-charged nano cerium oxide preferably comprises the following steps:
mixing an aqueous solution of cerium nitrate and an aqueous solution of polyacrylic acid to obtain a mixed solution;
and mixing the mixed solution with an ammonium hydroxide solution with the mass concentration of 30% for reaction to obtain the negatively charged nano cerium oxide.
In the present invention, it is preferable to mix an aqueous solution of cerium nitrate with an aqueous solution of polyacrylic acid to obtain a mixed solution. The concentration of cerium nitrate in the aqueous solution of cerium nitrate and the concentration of polyacrylic acid in the aqueous solution of polyacrylic acid are not particularly limited, and cerium nitrate and polyacrylic acid may be dissolved. In the invention, the mass ratio of the cerium nitrate to the polyacrylic acid is preferably 1-2:5-6.
After the mixed solution is obtained, the mixed solution is preferably mixed with an ammonium hydroxide solution with the mass concentration of 30% for reaction, so that the negatively charged nano cerium oxide is obtained. In the invention, the mass-volume ratio of the cerium nitrate to the ammonium hydroxide solution with the mass concentration of 30% is preferably 1-2 g/12-18 mL. In the present invention, the reaction time is preferably 20 to 28 hours, more preferably 24 hours; the temperature of the reaction is preferably 20 to 30 ℃, more preferably 23 to 28 ℃.
In the present invention, the application concentration of the negatively-charged nano-cerium oxide is preferably 0.05 to 0.1mM.
The technical solutions provided by the present invention are described in detail below in conjunction with examples for further illustrating the present invention, but they should not be construed as limiting the scope of the present invention.
Example 1
1. Preparation of negatively charged polyacrylic acid coated nano cerium oxide Particles (PNC)
1.08g of cerium nitrate (Sigma-Aldrich, 99%) and 4.5g of polyacrylic acid (1800 MW, sigma-Aldrich) were dissolved in 2.5mL (solution A) and 5.0mL (solution B) of deionized water, respectively. The A, B two solutions were thoroughly mixed using a vortex at 2000 rpm for 15min and the mixture was added drop-wise to a 50ml beaker containing 15ml of 30% ammonium hydroxide solution. The mixture was stirred at 500rpm for 24 hours at room temperature, then split into 1.5ml centrifuge tubes and centrifuged at 4000 g/min for 1 hour, the supernatant was aspirated and about 100 μl of liquid from the bottom of the tubes was discarded. The supernatant was aspirated into an ultrafiltration tube (MWCO 30K,Millipore Inc.) which was centrifuged at 4500rpm for 6 cycles (once every 45 minutes). After the cycle is completed, the absorption peak value of the solution is measured by a spectrophotometer, the concentration is calculated according to the specific law of Bell-Rainbow, and finally the solution is stored in a refrigerator at 4 ℃ for use.
2. Preparation of positively charged aminated cerium oxide nanoparticles (ANC)
3.5mL of 5mM PNC was mixed with 1.5mL of ultra pure water at 500rpm for 2 minutes at ambient temperature. Then, during further continuous stirring at 500rpm, 306.8mg of 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide (Sigma-Aldrich) (EDC) was dissolved in 0.5mL of MES buffer (100 mM, pH 6.0) and added dropwise to the mixture and stirred for 4 minutes. 800mM (6 mL) ethylenediamine (EDA, 99%, sigma-Aldrich) (pH 6.8 adjusted with HCl) was then added drop-wise to the final reaction mixture with continuous stirring at 500rpm, and stirring was continued for an additional 3 hours at ambient temperature. The resulting solution was transferred to a 1.5ml centrifuge tube and centrifuged at 4500rpm for 15 minutes to remove any debris and large clumps. The supernatant was purified by centrifugation at 4500rpm (Allegra X30, beckman) in 5 cycles (15 minutes each) using an ultrafiltration tube (MWCO 30K,Millipore Inc). After the cycle is completed, the absorption peak value of the solution is measured by a spectrophotometer, the concentration is calculated according to the specific law of Bell-Rainbow, and finally the solution is stored in a refrigerator at 4 ℃ for use.
3、CeO 2 NPs (Sigma-Aldrich) are commercial cerium oxide nanoparticles purchased from the company, which are labeled with particle size<25nm, the actual measurement particle size is about 60-70nm, and weak negative electricity.
4. Preparation of DiI-PNC
PNC was labeled with DiI (1, 1'-dioctadecyl-3, 3' tetramethyllindocarbonine, 1 '-octacosyl-3, 3' tetramethylindole carbocyanine, a fluorophore). 4ML,0.5mM PNC and 200. Mu.L, 0.3mg/mL DiI (dissolved in DMSO) were placed in a 20mL glass vial and mixed at 1000rpm for 1 minute. The resulting mixture was purified using a 10kDa filter (4500 rpm, 5min once, at least five times) to remove free chemicals. The final solution was labeled DiI-PNC and stored in a refrigerator at 4℃for further use.
PNC and CeO prepared by the method 2 The NPs were subjected to TEM detection, and the specific results are shown in FIG. 1. As can be seen from FIG. 1, the electron microscope results show that PNC has a particle size of less than 10nm and a regular elliptic shape, whereas CeO 2 NPs have large particle sizes and various shapes.
The characteristic absorption peaks of PNC, dil-PNC and ANC were measured by ultraviolet-visible spectrophotometry, respectively, and the specific results are shown in FIG. 2. As can be seen from FIG. 2, the PNC has a characteristic absorption peak of about 271nm, the ANC has an absorption peak of 264nm, and the Dil-labeled PNC has a characteristic absorption peak of 520nm and 570nm in addition to the PNC.
The PNC, the ANC and the CeO are respectively processed by adopting a laser particle sizer 2 The particle size of NPs was measured, and the specific results are shown in fig. 3. As can be seen from FIG. 3, PNC and ANC hydrate particlesThe diameter is below 10nm, ceO 2 The particle size of NPs was about 87nm.
For PNC, ANC and CeO respectively 2 The potential of NPs was measured, and the specific results are shown in FIG. 4. As can be seen from FIG. 4, PNC is shown with negative potential of about-40 mv, ANC is shown with positive potential of about +30mv, ceO 2 NPs show a weak negative potential of about-10 mv.
Example 2
Determination of Arabidopsis thaliana lateral root occurrence by PNC
1. Preparation of MS Medium
MS culture medium is prepared according to the proportion of 4.4g/L MS (Coolaber, beijing, china), 10g/L sucrose and 4g/L Phytagel (Coolaber, beijing, china), the PH is adjusted to 5.7-6.0, and the temperature is 121 ℃ and the temperature is high for 15min for sterilization. After sterilization, the MS medium was cooled to 60℃and 30ml of medium was fixed in a 50ml centrifuge tube (simultaneous sterilization).
The prepared MS culture medium is used as a control culture medium, PNC and CeCl are respectively added into the MS culture medium according to the application concentration of 0.1mM 3 ANC and CeO 2 The NPs nano material is used as an experimental group, poured into a sterile square culture dish with the volume of 10cm x 10cm after being uniformly mixed, and used after being cooled. All operations are performed in an ultra clean bench, with strict aseptic operations.
2. Arabidopsis seed disinfection, sowing and treatment
The Arabidopsis thaliana used in this patent is of the Col-0 type. The arabidopsis seeds are soaked, sucked and mixed for 10 seconds by 70% ethanol, washed for 2 times by sterile water, soaked by 5% NaClO and shaken up and down for 10 minutes, and washed for 3 times by sterile water. Uniformly broadcasting the seeds into MS square culture medium with 1ml gun head, blow-drying surface moisture, sealing with sealing film, vernalizing in dark environment at 4deg.C for 3 days, and placing under light (23 deg.C, 14/10h,200 μm) -2 s -1 ) Culturing for 5 days. After the arabidopsis seedlings grow for 5 days under the light, the arabidopsis seedlings with uniform growth vigor and root length are selected and respectively transferred to the culture medium of the experimental group and the culture medium of the control group (6 seedlings are placed on each plate, 4 plates are placed on the control group and the treatment group respectively), and vertically cultured for 7 days under the light, and the root phenotype map, the quantity of lateral roots and the dry fresh weight of the arabidopsis seedlings of the control group and the treatment group are counted, and the specific result is shown in figure 5. From the following componentsTable 5 shows that negatively charged cerium oxide nanoparticles PNC (-) are capable of promoting Arabidopsis lateral root initiation.
3. Root system active oxygen imaging of arabidopsis thaliana
Active oxygen in the root system of Arabidopsis thaliana was imaged using a laser confocal microscope. Using Dihydroethidium (DHE) and 2',7' -dichlorofluorescein diacetate (H) 2 DCFDA) as O respectively 2 - And H 2 O 2 Is a fluorescent dye of (a). Soaking root system of Arabidopsis grown for 7d on MS, PNC (0.1 mM) +MS medium in 25mu M H 2 DCFDA or 10 μm DHE dye (diluted with 10mM TES, ph=7.5) was incubated for 30min in dark conditions. After the incubation was completed, the slides were washed three times with TES and loaded with TES (a drop of Perfluoronaphthylamine (PFD) was previously added dropwise to the slide to enhance the fluorescence imaging effect), covered with coverslips, and ensured that there were no air bubbles. The confocal laser microscope was set as follows: a 40-fold objective lens, 488nm excitation light; PMT1:500nm-600nm; PMT2:700nm-800nm; h was calculated using Image J software with 4-6 replicates 2 DCFDA and DHE fluorescence intensities. The specific results are shown in fig. 6 and 7. As can be seen from fig. 6, fig. 6A is DHE (O is made 2 。- ) The staining chart, 6B is the fluorescence intensity value of DHE at the lateral root primordium, the higher the intensity, the higher the O 2 。- The more accumulated, the higher the DHE O was in stages 1-4 and in the lateral root development stage of PNC-treated lateral root primordia 2 。- Accumulation, whereas distribution in the primary roots is less than in the control group; FIG. 6C is O in root system as determined by the kit 2 。- Content, indicating O in the main and mature lateral roots after PNC treatment 2 。- Are all reduced. FIGS. 7A and 7B are DCF (H) 2 O 2 ) Staining pattern and DCF fluorescence intensity statistics, FIG. 7C shows H in root system measured by kit 2 O 2 Content, these data all indicate H in the root system of PNC-treated Arabidopsis 2 O 2 Is partially cleared.
Treatment of Arabidopsis thaliana with PNC at different concentrations
PNCs of 0.05, 0.07, 0.1, 0.15 and 0.2mM were added to MS medium, and Arabidopsis seedlings grown under light for 5 days (Arabidopsis seedlings with uniform growth vigor and root length were selected) were transferred to the above experiment medium and control medium (6 seedlings were placed on each plate, and 4 plates were placed on each control and treatment) respectively, and cultured vertically under light for 7 days, and the root phenotype map, root length and lateral root density of Arabidopsis seedlings of the control and treatment groups were counted, and the specific results are shown in FIG. 8. As can be seen from fig. 8, PNC was able to promote the lateral root development of arabidopsis seedlings, and the application concentration of 0.1Mm was most favorable for the lateral root development of arabidopsis.
PNC was localized in Arabidopsis root system using fluorescent-group-attached DiI-PNC (DiI-PNC was treated in the same manner as PNC addition). DiI-PNC was excited by 514nm laser, received at 550-615nm, and observed under laser confocal conditions, the specific results are shown in FIG. 9. After 24h of treatment of Arabidopsis thaliana by DiI-PNC, green fluorescence of DiI-PNC can be observed at root tip, indicating that PNC enters Arabidopsis thaliana root tip; after 48h of treatment of Arabidopsis thaliana with DiI-PNC, green fluorescence of DiI-PNC was observed at the lateral root primordia of the root system maturation zone, indicating that PNC entered the lateral root primordia.
Example 3
1. Rice seed disinfection sowing and treatment
The rice variety in this embodiment is quanyou 737. The rice seeds are treated with H with the mass concentration of 0.3% 2 O 2 Soaking, shaking at 50 rpm for 40 min, washing with clear water for 5min, air drying on absorbent paper, picking up full grains, placing seeds into germination box (germination box is 2ml centrifuge tube box with 100 holes, preferably covered with clear water, and allowing root system to grow), covering with cover, uncovering after 3 days, and placing under light (25deg.C, 14/10h,200 μm) -2 s -1 ) Culturing for 2 days to obtain 5-day-old rice seedlings. PNC treatment fluid was used to treat seedlings of 5 days old rice as the experimental group. Meanwhile, a control group (the control group is completely the same as the experimental group except that PNC is not added in the treatment liquid), and then rice seedlings with the size of 5 days are moved into a centrifuge tube under the light (25 ℃,14/10h,200 mu mol m) -2 s -1 ) Culturing, planting black field planting cotton, taking out after culturing for 6 days, and performing root indexThe statistics are shown in FIG. 11. As can be seen from FIG. 11, PNC was able to promote lateral root development of rice seedlings, and the applied concentration of 0.05Mm was most favorable for lateral root development.
The preparation method of the PNC treatment fluid specifically comprises the following steps: a15 ml brown centrifuge tube is used for filling rice nutrient solution, PNC is added to enable the nutrient solution to reach corresponding treatment concentration, and the formula of the business solution of the rice is shown in Table 1.
Table 1 Business liquid formula for rice
Example 4
1. Rape seed disinfection sowing and treatment
In this example, the rape seed is treated with H with a mass concentration of 0.3% in accordance with the rape seed type of Yangyou No. 9 2 O 2 Soaking, shaking at 50 rpm for 40 min, washing with clear water for 5min, air drying on absorbent paper, picking up full grains, placing seeds into germination box (germination box is 2ml centrifuge tube box with 100 holes, preferably covered with clear water, and allowing root system to grow), covering with cover, uncovering after 3 days, and placing under light (25deg.C, 14/10h,200 μm) -2 s -1 ) Culturing for 2 days to obtain 5-day-old rape seedlings. PNC treatment fluid was used to treat 5-day-old rape seedlings as an experimental group. Meanwhile, a control group (the control group is completely the same as the experimental group except that PNC is not added in the treatment liquid), rape seedlings with the size of 5 days are transferred into a centrifuge tube, and under the light (25 ℃,14/10h,200 mu mol m) -2 s -1 ) Culturing, planting black field planting cotton, taking out after culturing for 6 days, and counting root indexes, wherein the specific result is shown in figure 10. As can be seen from FIG. 10, PNC is capable of promoting the lateral root occurrence of rape seedlings, and the application concentration of 0.05Mm/L is most favorable for the lateral root occurrence.
The preparation method of the PNC treatment fluid specifically comprises the following steps: rape nutrient solution is filled into a 15ml brown centrifuge tube, PNC is added to achieve corresponding treatment concentration, and the formula of the rape business solution is shown in Table 2.
Table 2 formula of rape business liquid
The foregoing is merely a preferred embodiment of the present invention and it should be noted that modifications and adaptations to those skilled in the art may be made without departing from the principles of the present invention, which are intended to be comprehended within the scope of the present invention.
Claims (6)
1. The application of the nano cerium oxide in promoting the occurrence of plant lateral roots is characterized in that the nano cerium oxide is negatively charged nano cerium oxide; the granularity of the negatively charged nano cerium oxide is 1-10 nm.
2. The use according to claim 1, characterized in that the preparation method of the negatively-charged nano-cerium oxide comprises the following steps:
mixing an aqueous solution of cerium nitrate and an aqueous solution of polyacrylic acid to obtain a mixed solution;
and mixing the mixed solution with an ammonium hydroxide solution with the mass concentration of 30% for reaction to obtain the negatively charged nano cerium oxide.
3. The use according to claim 2, wherein the mass ratio of cerium nitrate to polyacrylic acid is 1-2:5-6.
4. The use according to claim 2, wherein the mass-to-volume ratio of cerium nitrate to ammonium hydroxide solution with a mass concentration of 30% is 1-2 g:12-18 ml.
5. The use according to claim 2, wherein the reaction time is 20-28 h and the temperature is 20-30 ℃.
6. The use according to claim 1, wherein the negatively-charged nano-ceria is applied at a concentration of 0.05-0.1 mm.
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