CN115836641A - Water culture seedling raising method for cruciferae hyper-enriched plants - Google Patents
Water culture seedling raising method for cruciferae hyper-enriched plants Download PDFInfo
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Abstract
The invention belongs to the technical field of seedling culture of hyper-enrichment plants, and particularly relates to a water culture seedling culture method of a cruciferous hyper-enrichment plant, which can effectively promote the germination of seeds and the growth of roots and improve the germination rate of the seeds by configuring an agar matrix containing nutrient elements such as nitrogen, phosphorus and potassium and hormones such as gibberellin, so that the germination rate of the seeds of the cruciferous hyper-enrichment plant is improved from 10-40% to 70%; the prepared water culture device is easy to transplant seedlings, does not damage roots, is beneficial to improving the survival rate of the seedlings from 30-60% to more than 80%, the cultured plant roots are adhered to the agar substrate and are easy to clean, the high cleanliness of the seedling roots is maintained, and the subsequent culture is not influenced; and the adopted nutrient solution reduces the content of phosphorus and is beneficial to the seedling cultivation of the cruciferous hyper-enriched plants.
Description
Technical Field
The invention belongs to the technical field of seedling culture of hyper-enriched plants, and particularly relates to a water culture seedling culture method of a hyper-enriched plant of cruciferae.
Background
According to survey bulletin of national soil pollution conditions, the total exceeding rate of national soil reaches 16.1%, the pollution type is mainly inorganic heavy metal pollution, the number of heavy metal exceeding points accounts for 82.8% of all exceeding points, and the method has potential environmental risks and needs to be repaired urgently.
The founder in the field of phytoremediation in 1983-the American Ministry of agriculture researcher Rufus Chaney, based on the discovery of hyper-enriched plants and the like, firstly proposed the concepts of phytoremediation by extraction and plant mining. The plant extraction technology is a novel remediation method for heavy metal pollution of soil, namely, super-enrichment plants are used for extracting heavy metal in the soil, and the method has the advantages of low cost, easiness in operation, no secondary pollution, small damage to the soil structure and the like. The method for extracting heavy metals rich in the harvested plants by further ashing and smelting the overground parts of the plants is called plant mining. However, at present, due to the lack of deep understanding of the heavy metal mechanism of hyper-enriched plants, the efficiency of plant extraction and plant mining cannot be effectively improved.
Therefore, a great deal of laboratory research work on the physiological mechanism of hyper-enriched plants is also carried out in the former, wherein a great deal of plant water culture work is involved, but some difficulties are encountered in the processes of seed germination and water culture, which mainly comprise: the seed germination efficiency in the common seedling culture medium is low, more seed germination and seedling culture mediums used at the present stage comprise common soil, peat soil, vermiculite, quartz sand, MS culture medium and the like, on the mediums, the seed germination rate of hyper-enriched plants such as Noccaea caerulescens (Thalassilia caerulea), odontarhena chalcidica (crambella denticulata) and Arabidopsis thaliana (Arabidopsis thaliana) in the culture medium is only 10-40%, and the seedling vigor of the germinated seedlings is weak; the germinated plant seedlings in the common seedling raising matrix are not easy to transfer, generally speaking, the hyper-enriched plants need to be transferred into a water culture nutrient solution for culture after being germinated for 7-10 days, but the germinated seedlings are not easy to separate from the common seedling raising matrix, and the roots are easy to be damaged in the separation process, so that the survival rate after seedling transfer is low; the roots of plant seedlings cultured by the common seedling culture medium are contaminated with more impurities, aseptic culture cannot be achieved, roots of plants growing in the common soil, peat soil and other mediums can be contaminated with the medium, effective cleaning cannot be achieved, and then subsequent detection of root system indexes is influenced.
Disclosure of Invention
In view of the above problems, the present invention aims to provide a water culture seedling method for cruciferae super-enriched plants, which is used for increasing the germination rate and survival rate of cruciferae super-enriched plants.
The technical content of the invention is as follows:
the invention provides a water culture seedling method of cruciferae hyper-enriched plants, which comprises the following steps:
1) Manufacturing a water culture device: taking a plastic pipe with two open ends, sealing one end of the plastic pipe with a sealing film to obtain a hydroponic pipe, and placing the hydroponic pipe on a device with holes to obtain a hydroponic device;
2) Preparing a seed germination matrix: preparing an agar substrate containing nitrogen, phosphorus, potassium and brassinosteroids;
the specific configuration method is as follows: mixing agar powder with water, adding potassium nitrate, potassium dihydrogen phosphate and calcium nitrate, boiling the obtained solution until agar is dissolved, sterilizing, autoclaving, adding hydrogen peroxide, cooling the agar solution to about 50 deg.C, adding brassinosteroid and manganese dioxide, stirring, packaging the obtained liquid matrix into water culture tubes, cooling, and solidifying to obtain seed germination matrix;
the addition of the manganese dioxide can accelerate the decomposition of the hydrogen peroxide, increase the oxygen content and the porosity of the matrix, ensure the complete removal of the hydrogen peroxide in the matrix and reduce the damage of the hydrogen peroxide to the root of the seed;
in the liquid matrix, the dosage of agar accounts for 5-8 wt% of the matrix, the concentration of potassium nitrate is 20-60 mM, the concentration of monopotassium phosphate is 2-6 mM, the concentration of calcium nitrate is 10-30 mM, the concentration of hydrogen peroxide is 0.1-0.3%, the dosage of brassinosterol is 0.3-1.0 mg/kg (L), and the dosage of manganese dioxide is 80-120mg/kg (L);
preferably, the concentration of potassium nitrate is 50mM, the concentration of monopotassium phosphate is 5mM, the concentration of calcium nitrate is 25mM, the concentration of hydrogen peroxide is 0.2%, the dosage of brassinosteroid is 0.5mg/kg, and the dosage of manganese dioxide is 100mg/kg;
3) Germination of seeds: the plant seeds are refrigerated in a refrigerator for 30 days, then disinfected by a 2 percent sodium hypochlorite solution for 30min, cleaned, soaked in a gibberellin solution (0.02-0.04 percent, preferably 0.03 percent) for 24h, cleaned, placed in a seed germination matrix, pressed into the matrix to a depth of about 3-5 mm, and placed in a humid environment to sprout in a dark place for 7-10 days;
the plant comprises a cruciferous hyperaccumulator seed, including Noccea caerulescens (Thalassia tenuifolia), odontarrha chalcidica (Thamnolia vermicularis) and Arabidopsis hellari (Arabidopsis thaliana);
4) Water planting of seedlings: after the seeds germinate and the seedlings grow to 2-3 cm, pre-culturing, removing a sealing film at the bottom of a water culture pipe, putting a water culture device in a water culture box filled with nutrient solution for culturing, firstly culturing for 6-7 days by adopting 25% of the nutrient solution, then culturing for 6-7 days by adopting 50% of the nutrient solution, and finally culturing by adopting 100% of the nutrient solution, wherein the seedlings can gradually adapt to a water culture environment through the culture mode, so that the growth of the seedlings is facilitated;
the nutrient solution comprises the components of H 3 BO 3 、MnCl 2 、CuSO 4 、Na 2 MoO 4 、Fe(III)-EDDHA、KNO 3 、KCl、KH 2 PO 4 、MgSO 4 、Ca(NO 3 ) 2 、Mg(NO 3 ) 2 、MES，pH5～6；
Furthermore, the content of each component in the nutrient solution is H 3 BO 3 20μM、MnCl 2 2μM、CuSO 4 0.4μM、Na 2 MoO 4 0.4μM、Fe(III)-EDDHA 20μM、KNO 3 2000μM、KCl 100μM、KH 2 PO 4 200μM、MgSO 4 1000μM、Ca(NO 3 ) 2 1000μM、Mg(NO 3 ) 2 500 mu M and MES 2000 mu M, and the dosage of each component in the nutrient solution is +/-20 percent based on the content.
The invention has the following beneficial effects:
the water culture seedling method of the cruciferae hyper-enriched plant is used for seed seedling of the cruciferae hyper-enriched plant, and can effectively promote the germination of seeds and the growth of roots and improve the germination rate of the seeds by configuring the oxygen-containing porous agar matrix containing nitrogen, phosphorus, potassium and other nutrient elements and brassinosteroid, so that the germination rate of the seeds of the cruciferae hyper-enriched plant is improved from 10-40% to 70%; the prepared water culture device is easy to transplant seedlings, does not damage roots, is beneficial to improving the survival rate of the seedlings from 30-60% to more than 80%, the cultured plant roots are adhered to the agar substrate and are easy to clean, the high cleanliness of the seedling roots is maintained, and the subsequent culture is not influenced; and the adopted nutrient solution reduces the content of phosphorus and is beneficial to the seedling cultivation of the cruciferae hyper-enrichment plant.
Drawings
FIG. 1 is a schematic view of a hydroponic apparatus as shown in example 1;
FIG. 2 is a schematic view of the hydroponic cassette of example 1.
Detailed Description
The present invention is described in further detail in the following description of specific embodiments and the accompanying drawings, it is to be understood that these embodiments are merely illustrative of the present invention and are not intended to limit the scope of the invention, which is defined by the appended claims, and modifications thereof by those skilled in the art after reading this disclosure that are equivalent to the above described embodiments.
All the raw materials and reagents of the invention are conventional market raw materials and reagents unless otherwise specified.
Example 1
Water culture seedling raising method for cruciferae hyper-enriched plants
1) Manufacturing a water culture device: cutting off 1/4 of the lower part and 1/3 of the upper part of a 1mL pipette tip to obtain a plastic pipe with two open ends (preferably, the plastic pipe is thick at the upper part and thin at the lower part, the lower nozzle angle is small, and agar is prevented from being missed), sealing the lower opening of the obtained middle part with a parafilm sealing film, as shown in the left drawing in the figure 1, obtaining a hydroponic pipe, then selecting a black KT plate with a proper size as required, drilling holes with the 1mL pipette tip at a distance of about 5cm, as shown in the right drawing in the figure 1, and placing the hydroponic pipe in the hole of the KT plate to obtain a hydroponic device;
2) Preparing a seed germination matrix: mixing 5g of agar powder with 1L of water, adding 5.055g of potassium nitrate, 0.68g of monopotassium phosphate and 4.1g of calcium nitrate, boiling the obtained solution until agar is dissolved, sterilizing, autoclaving for 15min at 121 ℃ by using an autoclave, adding 0.2% of hydrogen peroxide, adding 0.5mg of brassinosteroid and 100mg of manganese dioxide when the temperature of the agar solution is reduced to about 50 ℃, uniformly stirring, subpackaging the obtained liquid matrix into a water culture tube, and cooling and solidifying to obtain a seed germination matrix;
3) Germination of seeds: the method comprises the following steps of (1) refrigerating Noccaea caerulescens plant seeds in a refrigerator for 30 days, then disinfecting the Noccaea caerulescens plant seeds for 30min by using a 2% sodium hypochlorite solution, cleaning the Noccaea caerulescens plant seeds, soaking the Noccaea caerulescens plant seeds in a gibberellin solution (0.02-0.04%, optimally 0.03%) for 24h, cleaning the Noccaea caerulescens plant seeds, putting the Noccaea caerulescens plant seeds into a seed germination matrix, putting 1-2 seeds into the seed germination matrix, pressing the seeds into the matrix to a depth of about 3-5 mm, putting a water culture device into a humid environment, and germinating for 7-10 days in a dark place;
4) Water planting of seedlings: after the seeds germinate and the seedlings grow to 2-3 cm, pre-culturing, removing a parafilm sealing film at the bottom of a water culture pipe, placing a water culture device in a water culture box (shown in figure 2) filled with nutrient solution for culturing, firstly culturing for 7 days by adopting 25% of the nutrient solution, then culturing for 7 days by adopting 50% of the nutrient solution, and finally culturing for 7 days by adopting 100% of the nutrient solution, thus finishing the seedling culture;
the nutrient solution comprises the component H 3 BO 3 20μM、MnCl 2 2μM、CuSO 4 0.4μM、Na 2 MoO 4 0.4μM、Fe(III)-EDDHA 20μM、KNO 3 2000μM、KCl 100μM、KH 2 PO 4 200μM、MgSO 4 1000μM、Ca(NO 3 ) 2 1000μM、Mg(NO 3 ) 2 500 μ M MES 2000 μ M, pH 5.6.
Example 2
Water culture seedling raising method for cruciferae hyper-enriched plants
1) Manufacturing a water culture device: the same as example 1;
2) Preparing a seed germination matrix: mixing 6g of agar powder with 1L of water, adding 6.066g of potassium nitrate, 0.816g of monopotassium phosphate and 4.92g of calcium nitrate, boiling the obtained solution until agar is dissolved, sterilizing, autoclaving for 15min at 121 ℃ by using an autoclave, adding 0.3% of hydrogen peroxide, adding 1.0mg of brassinosteroid and 120mg of manganese dioxide when the temperature of the agar solution is reduced to about 50 ℃, uniformly stirring, subpackaging the obtained liquid matrix into a water culture tube, and cooling and solidifying to obtain a seed germination matrix;
3) Germination of seeds: storing Odontarhena chalicica plant seeds in a refrigerator for 30 days, disinfecting the seeds for 30min by using a 2% sodium hypochlorite solution, cleaning the seeds, soaking the seeds in a 0.04% gibberellin solution for 24h, cleaning the seeds, putting the seeds into a seed germination matrix, putting 1-2 seeds, pressing the seeds into the matrix to a depth of about 3-5 mm, placing a water culture device in a humid environment, and germinating in a dark place for 7-10 days;
4) Water planting of seedlings: after the seeds germinate and the seedlings grow to 2-3 cm, pre-culturing, removing a parafilm sealing film at the bottom of a water culture pipe, placing a water culture device in a water culture box filled with nutrient solution for culturing, firstly culturing for 7 days by adopting 25% of the nutrient solution, then culturing for 7 days by adopting 50% of the nutrient solution, and finally culturing for 7 days by adopting 100% of the nutrient solution, thus completing seedling culture;
the nutrient solution comprises the component H 3 BO 3 24μM、MnCl 2 2.4μM、CuSO 4 0.48μM、Na 2 MoO 4 0.48μM、Fe(III)-EDDHA 24μM、KNO 3 2400μM、KCl120μM、KH 2 PO 4 240μM、MgSO 4 1200μM、Ca(NO 3 ) 2 600μM、Mg(NO 3 ) 2 600 μ M MES 2400 μ M, pH 5.6.
Example 3
Water culture seedling raising method for cruciferae hyper-enriched plants
1) Manufacturing a water culture device: the same as example 1;
2) Preparing a seed germination matrix: mixing 8g of agar powder with 1L of water, adding 2.022g of potassium nitrate, 0.272g of monopotassium phosphate and 1.64g of calcium nitrate, boiling the obtained solution until agar is dissolved, sterilizing, autoclaving for 15min at 121 ℃ by using an autoclave, adding 0.1% of hydrogen peroxide, adding 0.3 mg of brassinosteroid and 80 mg of manganese dioxide when the temperature of the agar solution is reduced to about 50 ℃, uniformly stirring, subpackaging the obtained liquid matrix into a water culture tube, and cooling and solidifying to obtain a seed germination matrix;
3) Germination of seeds: storing the seeds of Arabidopsis hellari plants in a refrigerator for 30 days, then disinfecting the seeds for 30min by using a 2% sodium hypochlorite solution, cleaning the seeds, soaking the seeds in a 0.02% gibberellin solution for 24h, then cleaning the seeds, putting the seeds into a seed germination matrix, putting 1-2 seeds, pressing the seeds into the matrix to a depth of about 3-5 mm, placing a water culture device in a humid environment, and germinating in a dark place for 7-10 days;
4) Water culture of seedlings: after the seeds germinate and the seedlings grow to 2-3 cm, pre-culturing, removing a parafilm sealing film at the bottom of a water culture pipe, placing a water culture device in a water culture box filled with nutrient solution for culturing, firstly culturing for 6 days by adopting 25% of the nutrient solution, then culturing for 6 days by adopting 50% of the nutrient solution, and finally culturing for 7 days by adopting 100% of the nutrient solution, thus completing seedling culture;
the nutrient solution comprises the component H 3 BO 3 16 μM、MnCl 2 1.6 μM、CuSO 4 0.32μM、Na 2 MoO 4 0.32 μM、Fe(III)-EDDHA 16 μM、KNO 3 1600 μM、KCl80 μM、KH 2 PO 4 160 μM、MgSO 4 800 μM、Ca(NO 3 ) 2 800 μM、Mg(NO 3 ) 2 400. mu.M MES 1600. Mu.M, pH 5.6.
Comparative example 1
Soil seedling raising method for cruciferae hyper-enriched plants
1) The rice soil of Guangzhou south China agricultural university is adopted, and the main physicochemical properties are shown in the following table;
TABLE 1 physicochemical Properties of the soil
2) Sieving soil with 10 mesh sieve, mixing, placing into seedling box with soil depth of 4-6cm, and watering
Water is added to make the field water-holding rate reach 100%;
3) Soaking N.caerulescens plant seeds in 2% sodium hypochlorite solution for 30min, cleaning, soaking in 0.03% gibberellin solution for 24h, taking out, cleaning, uniformly sowing in a seedling box, covering 1 layer of soil with the thickness of about 1-2mm, and germinating in dark for 5-10 days;
4) And (3) seedling culture: after the seeds germinate and the seedlings grow to 2-3 cm, the seedlings are moved to a light culture room for pre-culture, the seedlings are taken out from the soil after 21 days, and the water culture is started after the roots are washed.
Comparative example 2
Seedling growing method for cruciferae hyper-enriched plants
1) An artificial seedling culture substrate is adopted, the main components of the artificial seedling culture substrate are 90% of moss peat and 10% of vermiculite, the pH value is 5.5-6.5, and the artificial seedling culture substrate contains 2.30% of nitrogen, 0.5% of phosphorus and 0.3% of potassium nutrient elements, so that the requirements of seed germination and early growth of seedlings can be met;
2) Placing the seedling substrate into a seedling box, wherein the depth of the substrate is about 4-6cm, and watering to moisten the substrate;
3) Soaking N.caerulescens plant seeds in 2% sodium hypochlorite solution for 30min, cleaning, soaking in 0.03% gibberellin solution for 24h, taking out, cleaning, uniformly sowing in a seedling box, covering 1 layer of matrix with thickness of about 1-2mm, and germinating in dark for 5-10 days;
4) And (3) seedling culture: after the seeds germinate and the seedlings grow to 2-3 cm, the seedlings are moved to a light culture room for pre-culture, the seedlings are taken out from the matrix after 21 days, and the seedlings are cleaned and subjected to water culture.
Comparative example 3
Odontarrhena chalicica, the procedure for growing seedlings was the same as in comparative example 2.
The seed germination and seedling growth of the cruciferous hyper-enriched plants in the seedling raising method of examples 1 to 3 and comparative examples 1 to 3 were recorded, and the results are shown below.
TABLE 2 seed Germination and seedling growth of cruciferous hyperaccumulator plants
As can be seen from Table 2, the cruciferae super-enriched plants treated by the embodiment of the invention have better seed germination and seedling growth conditions in comparison with each other, wherein the seed germination rates of different cruciferae super-enriched plants in the embodiments 1 to 3 are different. Compared with the proportion of 1-3, the oxygen-containing porous agar matrix containing nitrogen, phosphorus, potassium and other nutrient elements and brassinosteroids is prepared, so that the germination of seeds and the growth of roots can be effectively promoted, the germination rate of the seeds is improved, and the germination rate of the seeds of the cruciferous hyper-enriched plants is improved from 10-40% to 70%; the prepared water culture device is easy to transplant seedlings, does not damage roots, is beneficial to improving the survival rate of the seedlings from 30-60% to more than 80%, the cultured plant roots are adhered to the agar substrate and are easy to clean, the high cleanliness of the seedling roots is maintained, and the subsequent culture is not influenced; and the adopted nutrient solution reduces the content of phosphorus and is beneficial to the seedling cultivation of the cruciferae hyper-enrichment plant.
Claims (8)
1. A water culture seedling method of cruciferae hyper-enriched plants is characterized by comprising the following steps:
1) Manufacturing a water culture device: taking a plastic pipe with two open ends, sealing one end of the plastic pipe with a sealing film to obtain a hydroponic pipe, and placing the hydroponic pipe on a device with holes to obtain a hydroponic device;
2) Preparing a seed germination matrix: preparing a porous agar substrate containing nitrogen, phosphorus, potassium and brassinosteroids;
3) Germination of seeds: sterilizing plant seeds with 2% sodium hypochlorite solution, cleaning, soaking the plant seeds in gibberellin solution, cleaning, putting the plant seeds into a seed germination matrix, pressing the seeds into the matrix to a depth of about 3-5 mm, placing a water culture device in a humid environment, and germinating in a dark place for 7-10 days;
4) Water planting of seedlings: after the seeds germinate and the seedlings grow to 2-3 cm, pre-culturing, removing a sealing film at the bottom of a water culture pipe, putting a water culture device in a water culture box filled with nutrient solution for culturing, firstly culturing by adopting 25% of the nutrient solution, then culturing by adopting 50% of the nutrient solution, and finally culturing by adopting 100% of the nutrient solution.
2. The water culture seedling raising method for cruciferae super-enriched plants as claimed in claim 1, wherein the specific configuration method of the seed germination matrix in step 2) is as follows: mixing agar powder with water, adding potassium nitrate, potassium dihydrogen phosphate and calcium nitrate, boiling the obtained solution until agar is dissolved, sterilizing, autoclaving, and adding hydrogen peroxide; when the temperature of the agar solution is reduced to about 50 ℃, adding brassinosterol and manganese dioxide, uniformly stirring, subpackaging the obtained liquid matrix into a water culture pipe, and cooling and solidifying to obtain the seed germination matrix.
3. The method of claim 2, wherein agar accounts for 5-8 wt% of the substrate, potassium nitrate is 20-60 mM, potassium dihydrogen phosphate is 2-6 mM, calcium nitrate is 10-30 mM, hydrogen peroxide is 0.1-0.3%, brassinosteroid is 0.3-1.0 mg/kg (L), and manganese dioxide is 80-120mg/kg (L).
4. The method of claim 2, wherein agar is used in an amount of 5wt% of the substrate, potassium nitrate is 50mM, potassium dihydrogen phosphate is 5mM, calcium nitrate is 25mM, hydrogen peroxide is 0.2%, brassinosteroids is 0.5mg/kg, and manganese dioxide is 100mg/kg.
5. The hydroponic seedling method of cruciferous super-enriched plants as claimed in claim 1, wherein the plants of step 3) comprise cruciferous super-enriched plant seeds including Noccaea caerulescens, odontarhenia chalicica and Arabidopsis pishellari.
6. The hydroponic seedling method of the cruciferae super-enriched plant as claimed in claim 1, wherein the gibberellin solution of step 3) has a concentration of 0.02-0.04%.
7. The hydroponic seedling method for the cruciferae super-enriched plants as claimed in claim 1, wherein the component of the nutrient solution in step 4) comprises H 3 BO 3 、MnCl 2 、CuSO 4 、Na 2 MoO 4 、Fe(III)-EDDHA、KNO 3 、KCl、KH 2 PO 4 、MgSO 4 、Ca(NO 3 ) 2 、Mg(NO 3 ) 2 、MES，pH5～6。
8. The method of claim 7, wherein the plant is a hyper-enriched plant of BrassicaceaeThe water culture seedling raising method is characterized in that the content of each component in the nutrient solution is H 3 BO 3 20μM、MnCl 2 2μM、CuSO 4 0.4μM、Na 2 MoO 4 0.4μM、Fe(III)-EDDHA 20μM、KNO 3 2000μM、KCl 100μM、KH 2 PO 4 200μM、MgSO 4 1000μM、Ca(NO 3 ) 2 1000μM、Mg(NO 3 ) 2 500 mu M and MES 2000 mu M, and the dosage of each component in the nutrient solution is +/-20 percent based on the content.
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