Nicotiana benthamiana aerosol culture nutrient solution and tobacco cultivation method

Abstract

The present invention belongs to the technical field of soilless tobacco cultivation, and specifically relates to a nutrient solution for aeroponic cultivation of Nicotiana benthamiana, and a method for cultivating tobacco aeroponically using the nutrient solution. The present invention studies the cultivation methods of Nicotiana benthamiana aeroponics and proposes a nutrient solution formula suitable for the entire growth period of Nicotiana benthamiana, solving the problem of the lack of targeted nutrient solutions for soilless cultivation of Nicotiana benthamiana. Nicotiana benthamiana cultivated using this nutrient solution in an aeroponics device not only meets the nutritional needs of Nicotiana benthamiana during its growth and development, but also prevents the occurrence of various pests and diseases, effectively improving the growth state, quality, and yield of Nicotiana benthamiana, thus providing a relatively ideal aeroponic cultivation method for Nicotiana benthamiana.

Description

Technical Field

[0001] The invention belongs to the technical field of soilless tobacco cultivation, and particularly relates to a nutrient solution for aeroponic cultivation of Nicotiana benthamiana, and a cultivation method for aeroponic tobacco using the nutrient solution. Background Art

[0002] Nicotiana benthamiana belongs to the genus Nicotiana in the Solanaceae family. The plant is small but has large and lush leaves. It has the advantages of a short growth cycle and strong adaptability. Unlike the tobacco varieties used to make cigarettes, it has developed into a popular model system in plant biology due to its susceptibility to viruses and the widespread use of transient transgenic expression.

[0003] Julia Bally, Hyungtaek Jung and others studied the origin, spread, distribution, evolution, species and excellent traits of Nicotiana benthamiana that are widely used around the world, such as (1) it can be cultivated under laboratory conditions and is easy to grow and collect a large number of plant materials; (2) it has in-plant gene expression regulation and protein post-translational modification similar to model plants such as Arabidopsis thaliana and rice, which is more direct than functional verification in Escherichia coli or yeast expression systems; (3) it is sensitive to most known and identified plant viruses and can directly conduct plant-pathogen interaction function research; (4) the Agrobacterium-mediated transient gene expression system is mature, which can directly conduct molecular biology and biochemical function research such as gene regulation, protein interaction and localization.

[0004] Nicotiana benthamiana is an important model plant in current plant genetic engineering and breeding, and is the most widely used experimental host in plant virology. N. benthamiana is sensitive not only to viruses but also to many other pathogens, making it widely used in studies of plant and plant-pathogen interactions. Therefore, researching and developing a suitable cultivation model for N. benthamiana is of great biological significance.

[0005] Soil seeding, potting, and tissue culture techniques are common methods of tobacco production in my country. However, continuous cropping and repeated cropping in soil cultivation can lead to a decrease in soil fertility and the risk of soil-borne pests and diseases, which seriously impact tobacco yield, quality, and profitability. Potting is not conducive to large-scale, industrialized production and is time-consuming, labor-intensive, and costly. Tissue culture techniques are cumbersome and prone to contamination, resulting in unstable survival rates during hardening and transplanting, and difficult to control costs. In recent years, soilless cultivation has gained increasing attention in plant and crop production. Soilless cultivation uses a medium such as water, peat, vermiculite, perlite, and rockwool as a substrate to anchor the plant, with the plant roots directly exposed to the nutrient solution. Hydroponics involves immersing the plant roots in a nutrient solution, while aeroponics uses a spray device to atomize the nutrient solution into small droplets and spray them directly onto the plant roots.

[0006] Nutrient solution is the key to aeroponic cultivation. It contains macroelements and trace elements necessary for plant growth, and the proportions of various elements are appropriate. Otherwise, plant growth and development will be hindered, which will in turn affect the quality and yield of the crops. Different plants require different mineral elements, so different crops will have different nutrient solutions. Nicotiana benthamiana grown using aeroponic cultivation has the advantages of rapid growth and easy manual or mechanical movement for Agrobacterium infection. This cultivation model will be an important supporting technology for high-quality tobacco cultivation and production period regulation. Currently, there are relatively few studies on nutrient solutions for aeroponic tobacco cultivation, but there are certain patents and literature accumulations in the research on nutrient solutions for aeroponic cultivation of other crops. For example, the invention patent: CN 102295480 discloses a "Nutrient solution and cultivation method for greenhouse aeroponic potato microseed potatoes."

[0007] Currently, the nutrient solution used for growing Nicotiana benthamiana in greenhouses or growth rooms at research institutions and biotechnology companies across the country is mostly the universal Hoagland nutrient solution. However, Hoagland nutrient solution cultivation requires the addition of certain fertilizers, otherwise the plants are prone to excessive growth and malnutrition. Since Hoagland nutrient solution is universal and not a special nutrient solution for Nicotiana benthamiana, it cannot provide the nutrients needed for the tobacco's growth stage. Summary of the Invention

[0008] In order to address the defects of the prior art, the present invention provides a nutrient solution for aeroponic cultivation of Nicotiana benthamiana, and also provides a cultivation method for aeroponic cultivation of tobacco using the nutrient solution, providing data support for the large-scale production of Nicotiana benthamiana through soilless cultivation, enabling tobacco cultivation to be applied in plant factories.

[0009] The invention provides a Nicotiana benthamiana aerosol culture nutrient solution. The nutrient solution comprises a mother liquor A, a mother liquor B, and a mother liquor C. The mother liquor A comprises potassium nitrate and calcium nitrate tetrahydrate, uses pure water as a solvent, and the pH value of the solution is controlled between 3 and 4; the mother liquor B comprises potassium dihydrogen phosphate and magnesium sulfate heptahydrate, uses pure water as a solvent, and the pH value of the solution is controlled between 3 and 4; the mother liquor C comprises zinc sulfate heptahydrate, manganese sulfate monohydrate, sodium borate decahydrate, ammonium molybdate, and sodium ferric ethylenediaminetetraacetate, uses pure water as a solvent, and the pH value of the solution is controlled between 3 and 4.

[0010] Furthermore, in each 1L of Nicotiana benthamiana aerosol culture nutrient solution, the A mother solution contains the following components by molar mass:

[0011] <![CDATA[Potassium nitrate KNO3]]> 5.72~5.73mmol / L <![CDATA[Calcium nitrate tetrahydrate Ca(NO3)2·4H2O]]> 1.39~1.40mmol / L

[0012] Furthermore, in each 1 L of Nicotiana benthamiana aerosol culture nutrient solution, the B mother solution contains the following components by molar mass:

[0013] <![CDATA[Potassium dihydrogen phosphate KH2PO3]]> 1.52~1.53mmol / L <![CDATA[Magnesium sulfate heptahydrate MgSO4·7H2O]]> 0.96~0.97mmol / L

[0014] Furthermore, in every 1 L of Nicotiana benthamiana aerosol culture nutrient solution, the C mother solution contains the following components by molar mass:

[0015] <![CDATA[Zinc sulfate heptahydrate ZnSO4·7H2O]]> 0.016~0.017mmol / L <![CDATA[Manganese sulfate monohydrate MnSO4·H2O]]> 0.11~0.12mmol / L <![CDATA[Sodium tetraborate decahydrate Na2B4O7·10H2O]]> 0.015~0.016mmol / L <![CDATA[(NH4)2MOO4 ammonium molybdate]]> 0.001~0.002mmol / L <![CDATA[Sodium iron(III) ethylenediaminetetraacetate C 10 H 12 FeN2NaO8]]> 0.13~0.14mmol / L

[0016] The present invention also provides a method for preparing the Nicotiana benthamiana aerosol culture nutrient solution, comprising the following steps:

[0017] Step 1: Determine that the concentration multiples of the three mother liquors A, B, and C are 500, and the volumes of each are 1 L;

[0018] Step 2: Weigh 1 portion of each desired compound from mother liquor A according to the amount of 500-fold concentrated solution (potassium nitrate: 289 g, calcium nitrate tetrahydrate: 165 g) using an electronic scale;

[0019] Step 3: Place each compound in a beaker, dissolve it in purified water, and drain it into a 1L volumetric flask using a glass rod. Rinse each beaker 3-4 times with purified water, and drain the washing solution into a 1L volumetric flask using a glass rod.

[0020] Step 4: Add purified water to the mark, pour out the solution after it is fully dissolved, adjust the pH to 3-4 with sulfuric acid, then pour it into a 1L brown bottle, seal it and label it with the contents, preparation time, concentration multiple and other information, and store it in a dark place.

[0021] Step 5: Weigh 1 portion of each desired compound in mother liquor B according to the amount of 500-fold concentrated solution (magnesium sulfate heptahydrate: 118.835 g, potassium dihydrogen phosphate: 104 g) using an electronic scale;

[0022] Step 6: The preparation method of mother solution B is the same as steps 3 to 4 above;

[0023] Step 7: Weigh 1 portion of each desired compound from the C mother liquor (zinc sulfate heptahydrate: 2.43 g, manganese sulfate monohydrate: 9.82 g, sodium borate decahydrate: 2.55 g, ammonium molybdate: 0.145 g, sodium ferric EDTA: 28.57 g) using an electronic balance;

[0024] Step 8: Prepare Solution C in the same way as steps 3 to 4 above;

[0025] Step 9: Calculate the required volumes of the three mother solutions A, B, and C based on the required volume of the working solution. For example, after Nicotiana benthamiana is planted in the aeroponic device, nutrient solution needs to be added for cultivation. The device can accommodate 48L of nutrient solution, that is, the volume of the working solution is 48L. To dilute the three mother solutions 500 times, 96mL of mother solution A, 96mL of mother solution B, and 96mL of mother solution C are required.

[0026] Step 10: Measure 48L of tap water, then use a graduated cylinder to measure 96mL of A mother liquor, 96mL of B mother liquor, and 96mL of C mother liquor, and add them to the tap water in sequence, that is, add A mother liquor to dissolve first, then add B mother liquor to dissolve, and finally add C mother liquor to dissolve;

[0027] Step 11: Finally, measure and adjust the EC value and pH value of the nutrient solution.

[0028] In addition, the present invention also provides a method for cultivating Nicotiana benthamiana, which uses rock wool for tobacco breeding, plants tobacco on rock wool blocks, and then cultivates it with the above-mentioned Nicotiana benthamiana special nutrient solution; and uses a closed, fully artificial light vertical mist curtain plant aerosol cultivation device to grow tobacco.

[0029] The Nicotiana benthamiana cultivation method comprises the following steps:

[0030] Step S1: using rock wool to carry out tobacco breeding, using Nicotiana benthamiana aerosol culture nutrient solution to cultivate tobacco seedlings, wherein the nutrient solution for the tobacco seedling stage is the Nicotiana benthamiana aerosol culture nutrient solution, the EC value of the tobacco seedling stage nutrient solution is 1200-1400 μs / cm, and the pH is adjusted to 6.0;

[0031] Step S2: After 9 to 12 days of cultivation, the tobacco seedlings enter a fixed-value period and are transplanted to a closed, fully artificial light vertical mist curtain plant aeroponic cultivation device for cultivation. The nutrient solution used in the fixed-value period is the Nicotiana benthamiana aeroponic cultivation nutrient solution. The EC value of the nutrient solution in the fixed-value period is 1400 to 1600 μs / cm, and the pH is controlled between 5.4 and 6.8.

[0032] Step S3: After culturing for 6 to 8 days, the tobacco enters a vegetative growth phase. The nutrient solution during the vegetative growth phase is composed of a mixture of the Nicotiana benthamiana aerosol culture nutrient solution and D mother solution in a volume ratio of 3:1. The EC value of the nutrient solution during the vegetative growth phase is 1600 to 1900 μs / cm, and the pH is controlled between 5.4 and 6.8. The tobacco enters the vegetative growth phase and is continuously cultured for 8 to 10 weeks.

[0033] Furthermore, the Nicotiana benthamiana aerosol culture nutrient solution in step S1, step S2 and step S3 is prepared by successively adding mother liquor A, mother liquor B and mother liquor C in a volume ratio of 1:1:1 into water diluted 500 times and mixing them.

[0034] Furthermore, the preparation method of the D mother solution in step S3 is:

[0035] Collect the recovered nutrient solution from the tobacco nutrient solution during the fixed-value period, filter it, and obtain a filtrate. For every liter of filtrate, add 10 mL of citric acid, 5.5-8 mL of ABT stock solution 1.28, and 4 mL of pH Down reagent to prepare a D mother solution. The filter is a simple device assembled with a bottom layer of high-density water purification blanket ultrafine cotton and an upper layer of sponge. The pH of the D mother solution is 3-4.

[0036] Furthermore, the indoor environmental culture conditions of step S1, step S2 and step S3 are: the room temperature during the day is 22-25°C, and the room temperature at night is 19-22°C.

[0037] Furthermore, in step S2 and step S3, the internal temperature of the fog curtain is 19-24°C, the relative humidity of the indoor air is 70-87%, the photoperiod is 16 / 8h (light / dark), and the light intensity is 108-162 μmol / m 2 ·s.

[0038] Furthermore, in step S3, the temperature of the nutrient solution during the nutrient growth period is 19-23°C.

[0039] The tobacco nutrient solution provided herein has the same formula and preparation method for the seedling and transplanting stages, differing in the EC value of the nutrient solution. The EC value of the tobacco seedling-stage nutrient solution is lower than that of the transplanting-stage nutrient solution. Tobacco seedlings are cultured in seedling pots during the seedling stage and, after transplanting, are cultivated in a closed, fully artificial light, vertical mist curtain plant aeroponic cultivation device until ready for infection or seed harvesting. The nutrient solution for the vegetative growth stage is composed of a mixture of the N. benthamiana aeroponic nutrient solution and D mother liquor in a 3:1 volume ratio. Using this nutrient solution formula, and adjusting the EC value and pH value according to the needs of each N. benthamiana growth stage, the growth, quality, and yield of N. benthamiana can be effectively improved.

[0040] In addition, the pH of the nutrient solution in the Nicotiana benthamiana aerosol cultivation process provided by the present invention is controlled between 5.4 and 6.8 throughout the entire growth cycle. Tobacco root secretions will cause the pH of the nutrient solution to rise to about 8, and sulfuric acid is needed to adjust the pH of the nutrient solution, otherwise root rot will occur.

[0041] The nutrient solution for the aerosol cultivation process of Nicotiana benthamiana provided by the present invention is a nutrient solution specifically for Nicotiana benthamiana aerosol cultivation, which is prepared by successively adding mother liquor A, mother liquor B, and mother liquor C in a volume ratio of 1:1:1 to water with a certain dilution factor of 500 times, and can meet the nutritional requirements of Nicotiana benthamiana during its growth stage. The nutrient solution specifically for aerosol cultivation of Nicotiana benthamiana provided by the present invention is used from the time when true leaves appear and the tobacco reaches the cross stage after breeding. The EC value of the nutrient solution in the tobacco seedling stage is 1200-1400 μs / cm, and the pH is adjusted to about 6.0. The tobacco seedlings can be planted after being cultured in the nutrient solution for about 10 days, and are transplanted to a closed, fully artificial light, vertical mist curtain plant aerosol cultivation device for cultivation. The tobacco seedlings enter a fixed value period, which is a seedling acclimatization period, lasting about one week. The EC value of the nutrient solution in the fixed value period is 1400-1600 μs / cm. After the tobacco seedlings are acclimatized, the tobacco enters a vegetative growth period. The EC value of the nutrient solution in the vegetative growth period is 1600-1900 μs / cm. The nutrient solution used in the vegetative growth period is the nutrient solution specifically for aerosol cultivation of Nicotiana benthamiana added with mother liquor D. After 8 weeks of cultivation using this special nutrient solution for aeroponic Nicotiana benthamiana aerosol cultivation, compared with the Hoagland nutrient solution, the average plant height of Nicotiana benthamiana increased by 2.96 cm, the average maximum leaf length increased by 2.76 cm, and the average maximum leaf width increased by 1.56 cm, which can effectively promote the growth and development of Nicotiana benthamiana.

[0042] Compared to existing technologies, this invention explores aerosol cultivation methods for Nicotiana benthamiana and proposes a nutrient solution formula suitable for the entire Nicotiana benthamiana growth period, resolving the issue of untargeted nutrient solutions for soilless cultivation of Nicotiana benthamiana. Using this nutrient solution in an aerosol cultivation device, Nicotiana benthamiana not only meets its nutritional needs during growth and development but also protects against various pests and diseases, effectively improving its growth, quality, and yield. This represents an ideal aerosol cultivation method for Nicotiana benthamiana. BRIEF DESCRIPTION OF THE DRAWINGS

[0043] Figure 1 Schematic diagram of the culture and growth status of Nicotiana benthamiana in nutrient solution (Figure A is a schematic diagram of the growth of Nicotiana benthamiana seedlings planted in rock wool blocks, and Figure B is a schematic diagram of the growth status after planting and culturing with the nutrient solution prepared in Example 1 for 5 to 6 weeks);

[0044] Figure 2 Schematic diagram comparing the growth status of Nicotiana benthamiana cultured in two nutrient solutions after 8 weeks (Figure A is cultured in Hoagland nutrient solution, and Figure B is cultured in the nutrient solution prepared in Example 1 of the present invention);

[0045] Figure 3 This is the effect diagram of various growth indicators of Nicotiana benthamiana cultured with two nutrient solutions for 8 weeks;

[0046] Figure 4This is a growth status diagram of Nicotiana benthamiana planted in aerosol culture for 3 weeks after being cultured in Example 4;

[0047] Figure 5 The growth index results of Example 4 and Comparative Example 2 after 50 days of atomized cultivation (CK: Comparative Example 2-Hoagland nutrient solution, T1: nutrient solution prepared from Example 4 to Example 1 of the present invention);

[0048] Figure 6 The fresh weight index results of Example 4 and Comparative Example 2 after 50 days of atomization cultivation (CK: Comparative Example 2 ~ Hoagland nutrient solution, T1: Example 4 ~ nutrient solution prepared in Example 1 of the present invention);

[0049] Figure 7 The results of the number of leaves after 50 days of atomization cultivation in Example 4 and Comparative Example 2 (CK: Comparative Example 2 - Hoagland nutrient solution, T1: nutrient solution prepared in Example 4 - Example 1 of the present invention);

[0050] Figure 8 This is a curve diagram of the pH change of the Nicotiana benthamiana nutrient solution prepared in Example 1 (CK: pH change of Nicotiana benthamiana nutrient solution without adding D mother solution after 16 days of culture, T2: pH change of Nicotiana benthamiana nutrient solution after adding D mother solution after 16 days of culture). DETAILED DESCRIPTION

[0051] The present invention will be further described below by the description of specific embodiments, but this is not a limitation of the present invention. Those skilled in the art can make various modifications or improvements based on the basic idea of ​​the present invention, but as long as they do not depart from the basic idea of ​​the present invention, they are all within the scope of the present invention. The materials and reagents involved in the present invention can be obtained by commercially available or conventional technical means in the art. For example: pH Down reagent was purchased from Sichuan Yiheyuan Technology Co., Ltd., and ABT rooting powder was purchased from Beijing Aibidi Biotechnology Co., Ltd. (product name: ABT rooting powder No. 3).

[0052] Example 1: A Nicotiana benthamiana aerosol culture nutrient solution

[0053] The Nicotiana benthamiana aerosol culture nutrient solution is prepared by successively adding mother liquor A, mother liquor B, and mother liquor C into a certain amount of water in a volume ratio of 1:1:1 and mixing them together.

[0054] In 1 L of Nicotiana benthamiana aerosol culture nutrient solution, the A mother solution contains the following molar mass components:

[0055] <![CDATA[Potassium nitrate KNO3]]> 5.722mmol / L <![CDATA[Calcium nitrate tetrahydrate Ca(NO3)2·4H2O]]> 1.398mmol / L

[0056] In 1 L of Nicotiana benthamiana aerosol culture nutrient solution, the B mother solution contains the following molar mass components:

[0057] <![CDATA[Potassium dihydrogen phosphate KH2PO3]]> 1.528mmol / L <![CDATA[Magnesium sulfate heptahydrate MgSO4·7H2O]]> 0.965mmol / L

[0058] In 1 L of Nicotiana benthamiana aerosol culture nutrient solution, the C mother solution contains the following molar mass components:

[0059] <![CDATA[Zinc sulfate heptahydrate ZnSO4·7H2O]]> 0.0169mmol / L <![CDATA[Manganese sulfate monohydrate MnSO4·H2O]]> 0.116mmol / L <![CDATA[Sodium tetraborate decahydrate Na2B4O7·10H2O]]> 0.0157mmol / L <![CDATA[(NH4)2MOO4 ammonium molybdate]]> 0.0015mmol / L <![CDATA[Sodium iron(III) ethylenediaminetetraacetate C 10 H 12 FeN2NaO8]]> 0.130mmol / L

[0060] Method for preparing 48L nutrient solution:

[0061] Step 1: Determine that the concentration multiples of the three mother liquors A, B, and C are 500, and the volumes of each are 1 L;

[0062] Step 2: Weigh 1 portion of each desired compound from mother liquor A according to the amount of 500-fold concentrated solution (potassium nitrate: 289 g, calcium nitrate tetrahydrate: 165 g) using an electronic scale;

[0063] Step 3: Place each compound in a beaker, dissolve it in purified water, and drain it into a 1L volumetric flask using a glass rod. Rinse each beaker 3-4 times with purified water, and drain the washing solution into a 1L volumetric flask using a glass rod.

[0064] Step 4: Add purified water to the mark, pour out the solution after it is fully dissolved, adjust the pH to 3-4 with sulfuric acid, then pour it into a 1L brown bottle, seal it and label it with the contents, preparation time, concentration multiple and other information, and store it in a dark place.

[0065] Step 5: Weigh 1 portion of each desired compound in mother liquor B according to the amount of 500-fold concentrated solution (magnesium sulfate heptahydrate: 118.835 g, potassium dihydrogen phosphate: 104 g) using an electronic scale;

[0066] Step 6: The preparation method of mother solution B is the same as steps 3 to 4 above;

[0067] Step 7: Weigh 1 portion of each desired compound from the C mother liquor (zinc sulfate heptahydrate: 2.43 g, manganese sulfate monohydrate: 9.82 g, sodium borate decahydrate: 2.55 g, ammonium molybdate: 0.145 g, sodium ferric EDTA: 28.57 g) using an electronic balance;

[0068] Step 8: Prepare Solution C in the same way as steps 3 to 4 above;

[0069] Step 9: Calculate the required volumes of the three mother solutions A, B, and C based on the required volume of the working solution. For example, after tobacco is planted in the aeroponic device, nutrient solution needs to be added for cultivation. The device can accommodate 48L of nutrient solution, that is, the volume of the working solution is 48L. To dilute the three mother solutions 500 times, 96mL of mother solution A, 96mL of mother solution B, and 96mL of mother solution C are required.

[0070] Step 10: Measure 48L of tap water, then use a graduated cylinder to measure 96mL of A mother liquor, 96mL of B mother liquor, and 96mL of C mother liquor, and add them to the tap water in sequence, that is, add A mother liquor to dissolve first, then add B mother liquor to dissolve, and finally add C mother liquor to dissolve;

[0071] Step 11: Finally, measure and adjust the EC value and pH value of the nutrient solution.

[0072] Example 2: A Nicotiana benthamiana aerosol culture nutrient solution

[0073] The Nicotiana benthamiana aerosol culture nutrient solution is prepared by successively adding mother liquor A, mother liquor B, and mother liquor C into a certain amount of water in a volume ratio of 1:1:1 and mixing them together.

[0074] In 1 L of Nicotiana benthamiana aerosol culture nutrient solution, the A mother solution contains the following molar mass components:

[0075] <![CDATA[Potassium nitrate KNO3]]> 5.722mmol / L <![CDATA[Calcium nitrate tetrahydrate Ca(NO3)2·4H2O]]> 1.398mmol / L

[0076] In 1 L of Nicotiana benthamiana aerosol culture nutrient solution, the B mother solution contains the following molar mass components:

[0077] <![CDATA[Potassium dihydrogen phosphate KH2PO3]]> 1.528mmol / L <![CDATA[Magnesium sulfate heptahydrate MgSO4·7H2O]]> 0.965mmol / L

[0078] In 1 L of Nicotiana benthamiana aerosol culture nutrient solution, the C mother solution contains the following molar mass components:

[0079] <![CDATA[Zinc sulfate heptahydrate ZnSO4·7H2O]]> 0.0169mmol / L <![CDATA[Manganese sulfate monohydrate MnSO4·H2O]]> 0.116mmol / L <![CDATA[Sodium tetraborate decahydrate Na2B4O7·10H2O]]> 0.0157mmol / L <![CDATA[(NH4)2MOO4 ammonium molybdate]]> 0.0015mmol / L <![CDATA[Sodium iron(III) ethylenediaminetetraacetate C 10 H 12 FeN2NaO8]]> 0.130mmol / L

[0080] Method for preparing 12L nutrient solution:

[0081] Step 1: Determine that the concentration multiples of the three mother liquors A, B, and C are 500, and the volumes of each are 1 L;

[0082] Step 2: Weigh 1 portion of each desired compound from mother liquor A according to the amount of 500-fold concentrated solution (potassium nitrate: 289 g, calcium nitrate tetrahydrate: 165 g) using an electronic scale;

[0083] Step 3: Place each compound in a beaker, dissolve it in purified water, and drain it into a 1L volumetric flask using a glass rod. Rinse each beaker 3-4 times with purified water, and drain the washing solution into a 1L volumetric flask using a glass rod.

[0084] Step 4: Add purified water to the mark, pour out the solution after it is fully dissolved, adjust the pH to 3-4 with sulfuric acid, then pour it into a 1L brown bottle, seal it and label it with the contents, preparation time, concentration multiple and other information, and store it in a dark place.

[0085] Step 5: Weigh 1 portion of each desired compound in mother liquor B according to the amount of 500-fold concentrated solution (magnesium sulfate heptahydrate: 118.835 g, potassium dihydrogen phosphate: 104 g) using an electronic scale;

[0086] Step 6: The preparation method of mother solution B is the same as steps 3 to 4 above;

[0087] Step 7: Weigh 1 portion of each desired compound from the C mother liquor (zinc sulfate heptahydrate: 2.43 g, manganese sulfate monohydrate: 9.82 g, sodium borate decahydrate: 2.55 g, ammonium molybdate: 0.145 g, sodium ferric EDTA: 28.57 g) using an electronic balance;

[0088] Step 8: Prepare Solution C in the same way as steps 3 to 4 above;

[0089] Step 9: Calculate the required volumes of the three mother solutions A, B, and C based on the required volume of the working solution. For example, after tobacco is planted in the aeroponic device, nutrient solution needs to be added for cultivation. The device can hold 12L of nutrient solution, which means the volume of the working solution is 12L. To dilute the three mother solutions 500 times, 24mL of mother solution A, 24mL of mother solution B, and 24mL of mother solution C are required.

[0090] Step 10: Measure 12L of tap water, then use a graduated cylinder to measure 24mL of A mother liquor, 24mL of B mother liquor, and 24mL of C mother liquor, and add them to the tap water in sequence, that is, first add A mother liquor to dissolve, then add B mother liquor to dissolve, and finally add C mother liquor to dissolve;

[0091] Step 11: Finally, measure and adjust the EC value and pH value of the nutrient solution.

[0092] Example 3: Rockwool Block Hydroponic Planting Method for Nicotiana benthamiana

[0093] Soak the Nicotiana benthamiana seeds in pure water for 4 hours, then use tweezers to place them in the depression of the seedling plug for germination. The cross tobacco seedlings are cultured with the nutrient solution prepared in Example 1, and the EC value of the nutrient solution is 1300 μs / cm. When the Nicotiana benthamiana grows 4 to 5 true leaves, the strong seedlings are selected and transplanted into rock wool blocks for cultivation. The nutrient solution prepared in Example 1 is used for cultivation throughout the growth period, and the EC value of the nutrient solution is 1600 μs / cm and the pH is 6.3. The rock wool blocks are cultured for 8 weeks to ensure that they are moist. A thin layer of nutrient solution is spread on the bottom of the water tank. After the bottom of the water tank is dry, another thin layer of nutrient solution is spread for soaking.

[0094] The growth of the Nicotiana benthamiana rock wool block hydroponic planting of Example 3 of the present invention after 8 weeks is shown in the figure. Figure 1 As shown, Figure A is a schematic diagram of the growth of Nicotiana benthamiana seedlings planted in rock wool blocks, and Figure B is a schematic diagram of the growth status of Nicotiana benthamiana seedlings after being cultured in the nutrient solution prepared in Example 1 for 5 to 6 weeks after planting.

[0095] Comparative Example 1: Rockwool Block Hydroponic Planting Method for Nicotiana benthamiana

[0096] Soak Nicotiana benthamiana seeds in pure water for 4 hours, then use tweezers to place them in the depression of the seedling plug for germination. Use Hoagland nutrient solution for culture during the seedling stage of Nicotiana benthamiana, and the EC value of the Hoagland nutrient solution is 1300 μs / cm. When Nicotiana benthamiana grows 4 to 5 true leaves, screen out strong seedlings and transplant them into rock wool blocks for culture. Use Hoagland nutrient solution for culture throughout the growth period, and the EC value of the Hoagland nutrient solution is 1600 μs / cm and the pH is 6.3. Culture for 8 weeks to ensure that the rock wool blocks are moist. Spread a thin layer of nutrient solution on the bottom of the sink. After the bottom of the sink is dry, spread another thin layer of nutrient solution for soaking.

[0097] The components of the Hoagland nutrient solution are shown in Table 1:

[0098]

[0099]

[0100] Example 4: Atomization Cultivation Method for Nicotiana benthamiana

[0101] Rock wool was used for tobacco breeding. Tobacco was planted on rock wool blocks and then cultured with the nutrient solution prepared in Example 1. Tobacco was planted using a closed, fully artificial light, vertical mist curtain, and plant aerosol cultivation device.

[0102] The Nicotiana benthamiana cultivation method comprises the following steps:

[0103] Step S1: soaking Nicotiana benthamiana seeds in purified water for 4 hours, then placing them in the depression of a seedling plug with tweezers for germination, using rock wool for tobacco breeding, and using a Nicotiana benthamiana aeroponic nutrient solution to cultivate tobacco seedlings. The nutrient solution for the tobacco seedling stage is the tobacco aeroponic nutrient solution prepared in Example 1, and the EC value of the tobacco seedling stage nutrient solution is 1300 μs / cm, and the pH is adjusted to 6.0. The culturing conditions in the indoor environment are: daytime room temperature of 24° C. and nighttime room temperature of 22° C.

[0104] Step S2: After culturing the tobacco seedlings for 10 days, the seedlings enter a fixed-value period. When the tobacco grows 4 to 5 true leaves, the strong seedlings are selected and placed in a planting cup. The seedlings are then transplanted to a closed, fully artificial light, vertical mist curtain, plant aeroponic cultivation device for cultivation. The nutrient solution used in the fixed-value period is the tobacco aeroponic cultivation nutrient solution prepared in Example 1. The EC value of the nutrient solution in the fixed-value period is 1500 μs / cm, and the pH is controlled at 6.3.

[0105] Step S3: After culturing for 6 days, the tobacco enters a vegetative growth phase. The nutrient solution in the vegetative growth phase is composed of a mixture of the tobacco aerosol culture nutrient solution prepared in Example 1 and D mother solution in a volume ratio of 3:1. The EC value of the nutrient solution in the vegetative growth phase is 1800 μS / cm, and the pH is controlled at 6.4. The tobacco enters the vegetative growth phase and is cultured for 8 weeks.

[0106] The culture conditions of the indoor environment in step S2 and step S3 are: the room temperature during the day is 24°C and the room temperature at night is 22°C; the temperature inside the fog curtain in step S2 and step S3 is 22°C, the relative humidity of the indoor air is 80%, the photoperiod is 16 / 8h (light / dark), and the light intensity is 150μmol / m 2 ·s; the temperature of the nutrient solution during the nutrient growth period in step S3 is 20°C;

[0107] The preparation method of the D mother solution in step S3 is:

[0108] Collect the tobacco fixed-value period nutrient solution recovery liquid, filter to obtain the filtrate, add 10mL citric acid, 6mL ABT stock solution 1.28, and 4mL pH Down reagent to every 1L of filtrate to prepare D mother solution, and the pH of the D mother solution is 3-4.

[0109] The growth status of tobacco aerosol cultured for 3 weeks after planting in Example 4 of the present invention is shown in FIG. Figure 4 shown.

[0110] Comparative Example 2: Atomization Cultivation Method of Nicotiana benthamiana

[0111] Tobacco is bred using rock wool, planted on rock wool blocks, and then cultured with Hoagland nutrient solution; tobacco is grown using a closed, fully artificial light, vertical mist curtain, and plant aerosol cultivation device.

[0112] The Nicotiana benthamiana cultivation method comprises the following steps:

[0113] Step S1: soaking Nicotiana benthamiana seeds in purified water for 4 hours, then placing them in the depression of a seedling plug with tweezers for germination, using rock wool for tobacco breeding, and using Hoagland nutrient solution for culturing tobacco seedlings. The Hoagland nutrient solution has an EC value of 1300 μs / cm and a pH adjusted to 6.0. The culturing conditions in the indoor environment are: a room temperature of 24° C. during the day and 22° C. at night.

[0114] Step S2: After culturing the tobacco seedlings for 10 days, the seedlings enter a fixed value period. When the tobacco seedlings grow 4 to 5 true leaves, strong seedlings are selected and placed in planting cups. The seedlings are then transplanted to a closed, fully artificial light vertical mist curtain plant aeroponic cultivation device for cultivation. The nutrient solution used during the fixed value period is Hoagland nutrient solution, the EC value of the Hoagland nutrient solution is 1500 μs / cm, and the pH is controlled at 6.3.

[0115] Step S3: After culturing for 6 days, the tobacco enters a vegetative growth phase. The nutrient solution used in the vegetative growth phase is a Hoagland nutrient solution having an EC value of 1800 μS / cm and a pH value of 6.4. The tobacco is cultured for 8 weeks in the vegetative growth phase.

[0116] The culture conditions of the indoor environment in step S2 and step S3 are: the room temperature during the day is 24°C and the room temperature at night is 22°C; the temperature inside the fog curtain in step S2 and step S3 is 22°C, the relative humidity of the indoor air is 80%, the photoperiod is 16 / 8h (light / dark), and the light intensity is 150μmol / m 2 ·s; the temperature of the nutrient solution during the nutrient growth period in step S3 is 20°C.

[0117] The components of the Hoagland nutrient solution are shown in Table 1 in Comparative Example 1.

[0118] Test Example 1: Comparative Growth Test of Nicotiana benthamiana Planted in Rockwool Blocks Hydroponically

[0119] 1. Test subjects:

[0120] Nicotiana benthamiana grown hydroponically with rock wool blocks in Example 3 and Comparative Example 1.

[0121] 2. Test method:

[0122] The growth status of Nicotiana benthamiana plants in Example 3 and Comparative Example 1 after 8 weeks of cultivation was observed and photographed. The plant height, leaf length, and leaf width of Nicotiana benthamiana plants in Example 3 and Comparative Example 1 after 8 weeks of cultivation were also measured. The plant height was measured with a long ruler, the maximum leaf length was the maximum leaf length measured by the ruler within the entire tobacco plant leaf, and the maximum leaf width was the maximum leaf width measured by the ruler within the entire tobacco plant leaf.

[0123] 3. Test results:

[0124] 3.1. Comparative diagram of the growth status of Nicotiana benthamiana after 8 weeks of cultivation in Example 3 and Comparative Example 1. Figure 2 shown.

[0125] 3.2. The test results of plant height, leaf length and leaf width of Nicotiana benthamiana after 8 weeks of cultivation of Example 3 and Comparative Example 1 are shown in Table 2 and Figure 3 shown.

[0126] Table 2 Test data of plant height, leaf length and leaf width of Nicotiana benthamiana after 8 weeks

[0127] Plant height (cm) Maximum leaf length (cm) Maximum leaf width (cm) Example 3 23.54 15.52 11.28 Comparative Example 1 20.58 12.76 9.72

[0128] As can be seen from Table 2, the plant height, maximum leaf length, and maximum leaf width of Nicotiana benthamiana grown hydroponically using the rock wool blocks containing the nutrient solution prepared in Example 1 of the present invention are greater than those grown using the Hoagland nutrient solution. The plant height of Nicotiana benthamiana increased by an average of 2.96 cm, the maximum leaf length increased by an average of 2.76 cm, and the maximum leaf width increased by an average of 1.56 cm, indicating that the nutrient solution formula provided by the present invention is more capable of promoting the growth of Nicotiana benthamiana in terms of indicators such as plant height, leaf length, and leaf width.

[0129] Test Example 2: Comparative Growth Test of Nicotiana benthamiana in Atomized Cultivation

[0130] 1. Test subjects:

[0131] Nicotiana benthamiana cultivated by atomization in Example 4 and Comparative Example 2.

[0132] 2. Test method:

[0133] In the two aeroponic devices of Example 4 and Comparative Example 2, Nicotiana benthamiana was cultured using Hoagland nutrient solution and the nutrient solution prepared in Example 1 of the present invention, respectively. The breeding, transplanting, and cultivation times of Nicotiana benthamiana in the two devices were consistent. On the 29th, 35th, 44th, and 50th days after the start of breeding, 9 Nicotiana benthamiana plants were randomly selected from the aeroponic device for measurement of plant height, root length, fresh weight, and leaf number. After measurement, the plants were marked and returned to their original positions for continued cultivation. After the 50th day of measurement, the average plant height, root length, fresh weight, and leaf number of the 9 tobacco plants were taken to plot a growth curve. Plant height and root length were measured with a ruler, fresh weight was weighed with an electronic scale, and leaf number was directly visually measured. At the same time, after the tobacco seedlings were planted in the aeroponic device, the pH value of the nutrient solution was measured every 3 days. After 16 days of planting, the pH value of the nutrient solution was measured every 3 days with and without the addition of D mother solution. The pH of the nutrient solution was measured using a pH meter.

[0134] 3. Test results:

[0135] 3.1. The growth results of Example 4 and Comparative Example 2 after 50 days of atomization cultivation are shown in Tables 3 to 5. Figures 5 to 7 As shown:

[0136] (1) The growth index results of Example 4 and Comparative Example 2 after atomization cultivation for 50 days are shown in Table 3 and Figure 5 As shown:

[0137] Table 3 Growth index test data of Example 4 and Comparative Example 2 after 50 days of atomization cultivation

[0138]

[0139] (2) The fresh weight index results of Example 4 and Comparative Example 2 after atomization cultivation for 50 days are shown in Table 4 Figure 6 As shown:

[0140] Table 4 Fresh weight index test data of Example 4 and Comparative Example 2 after 50 days of atomization cultivation

[0141] Time days Example 4-T1(g) Comparative Example 2-CK (g) 29 days 7.50 5.30 35 days 7.96 6.89 44 days 13.86 12.88 50 days 15.73 14.30

[0142] (3) The leaf counts of Example 4 and Comparative Example 2 after 50 days of atomization cultivation are shown in Tables 5 and Figure 7 As shown:

[0143] Table 5 Experimental data of leaf number of Example 4 and Comparative Example 2 after 50-day atomization cultivation

[0144]

[0145]

[0146] As can be seen from Tables 3 to 5, the plant height, root length, fresh weight, and leaf number of Nicotiana benthamiana cultivated using the nutrient solution prepared in Example 1 of the present invention are greater than those of Nicotiana benthamiana cultivated using the Hoagland nutrient solution. This indicates that the Nicotiana benthamiana nutrient solution provided by the present invention is more suitable for the growth and development of Nicotiana benthamiana than the Hoagland nutrient solution, and improves the growth quality of Nicotiana benthamiana.

[0147] 3.2. The pH value change curve of the Nicotiana benthamiana nutrient solution prepared in Example 1 is as follows: Figure 8 As shown:

[0148] Tobacco root secretions will cause the pH of the nutrient solution to rise to about 8, otherwise root rot will occur. Figure 8 It can be seen that the pH of the Nicotiana benthamiana nutrient solution prepared by the present invention is always maintained between 5.4 and 6.8, which reduces the probability of Nicotiana benthamiana rot and the number of times the pH of Nicotiana benthamiana is adjusted. This not only more effectively promotes the growth and development of Nicotiana benthamiana, but also simplifies the work process. It is a more ideal Nicotiana benthamiana atomized culture nutrient solution.

[0149] The above embodiments are merely illustrative of the principles and effects of the present invention and are not intended to limit the present invention. Anyone skilled in the art may modify or alter the above embodiments without departing from the spirit and scope of the present invention. Therefore, all equivalent modifications or alterations made by one of ordinary skill in the art without departing from the spirit and technical principles disclosed herein are intended to be covered by the claims of the present invention.

Claims

1. A method for cultivating Nicotiana benthamiana, characterized in that: The following steps are involved: Step S1: using rock wool to breed tobacco, using Nicotiana benthamiana aerosol culture nutrient solution to culture tobacco seedlings, wherein the EC value of the tobacco seedling nutrient solution is 1200-1400 μs / cm, and the pH is adjusted to 6.0; Step S2: After 9 to 12 days of cultivation, the tobacco seedlings enter the planting period and are transplanted to a closed, fully artificial light vertical mist curtain plant aeroponic cultivation device for cultivation. The nutrient solution used during the planting period is a Nicotiana benthamiana aeroponic cultivation nutrient solution. The EC value of the nutrient solution during the planting period is 1400 to 1600 μs / cm, and the pH is controlled between 5.4 and 6.

8. Step S3: After 6 to 8 days of cultivation, the tobacco enters a vegetative growth phase. The nutrient solution during the vegetative growth phase is a mixture of Nicotiana benthamiana aeroponic nutrient solution and D mother solution in a volume ratio of 3:

1. The EC value of the nutrient solution during the vegetative growth phase is 1600 to 1900 μs / cm, and the pH is controlled between 5.4 and 6.

8. The tobacco enters the vegetative growth phase and is continuously cultivated for 8 to 10 weeks. The aerosol culture nutrient solution for Nicotiana benthamiana is prepared by successively adding mother liquor A, mother liquor B, and mother liquor C in a volume ratio of 1:1:1 into water diluted 500 times, wherein per 1L of the nutrient solution, the mother liquor A contains the following components by molar mass: 5.72-5.73 mmol / L of potassium nitrate and 1.39-1.40 mmol / L of calcium nitrate tetrahydrate; and per 1L of the nutrient solution, the mother liquor B contains the following components by molar mass: 1.52-1.53 ​​mmol / L of potassium dihydrogen phosphate. ol / L, magnesium sulfate heptahydrate 0.96-0.97mmol / L; in each 1L of nutrient solution, the C mother solution contains the following molar mass components: zinc sulfate heptahydrate 0.016-0.017mmol / L, manganese sulfate monohydrate 0.11-0.12mmol / L, sodium borate decahydrate 0.015-0.016mmol / L, ammonium molybdate 0.001-0.002mmol / L, and sodium ferric ethylenediaminetetraacetate 0.13-0.14mmol / L; The preparation method of the D mother solution is: Collect the recovered nutrient solution during the tobacco planting period, filter it to obtain the filtrate, and add 10 mL of citric acid, 5.5-8 mL of ABT stock solution 1.28, and 4 mL of pH Down reagent to every 1 L of the filtrate to prepare the D mother solution.

2. The method for cultivating Nicotiana benthamiana according to claim 1, wherein: The indoor environmental culture conditions of step S1, step S2 and step S3 are: the room temperature during the day is 22-25° C., and the room temperature at night is 19-22° C.

3. The method for cultivating Nicotiana benthamiana according to claim 1, wherein: In the steps S2 and S3, the internal temperature of the fog curtain is 19-24° C., the relative humidity of the indoor air is 70-87%, the photoperiod is 16 / 8h (light / dark), and the light intensity is 108-162 μmol / m 2 ·s.

4. The method for cultivating Nicotiana benthamiana according to claim 1, wherein: The temperature of the nutrient solution during the nutrient growth period in step S3 is 19-23°C.

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