A method for preparing composite pollen based on carbon nanopowder

Abstract

The invention belongs to the technical field of hybrid seed production, and particularly relates to a method for preparing composite pollen based on carbon nanopowder; the method comprises the following steps: S1, dissolving active ingredients to prepare a solution of a specific concentration; S2, weighing carbon nanopowder, and adding the carbon nanopowder to the solution prepared in step S1 according to a proportion; S3, fully stirring until the carbon nanopowder and the liquid are evenly mixed; S4, placing the mixture in an oven with air blowing at 30°C to air-dry the liquid on the surface of the mixture, adjusting the temperature to 40-50°C, drying, and agglomerating the solid into blocks; S5, grinding the agglomerated carbon powder solids into powder in a mortar; S6, fully and evenly mixing the active powder with pure pollen according to a proportion, sealing and storing, and dispersing a specific mass of quercetin on the carbon nanoparticles to the greatest extent, thereby improving the promoting effect of non-water-soluble quercetin on pollen, improving the pollen germination rate, and reducing the amount of pollen used.

Description

Technical Field

[0001] The invention belongs to the technical field of hybrid seed production, and particularly relates to a method for preparing composite pollen based on carbon nanopowder. Background Art

[0002] In the sexual reproduction process of plants, pollen, as a germplasm resource, plays a crucial role and is the most fundamental material for hybrid breeding and seed propagation. To protect the intellectual property rights of varieties, hybrid seed production or male sterile seed production has become a trend in tobacco seed production. Major domestic tobacco varieties such as Yunyan 87 and K326 have been bred into male sterile lines. However, the reproduction and production of male sterile and hybrid seeds requires pollination, particularly artificial pollination, to achieve optimal production efficiency. In tobacco hybrid seed production and male sterile seed propagation, due to tobacco's long flowering period and the small and numerous floral organs such as stamens and pistils, pollen viability is significantly affected by collection quality, production, and storage environment. This makes pollen collection and pollination extremely labor-intensive and costly. Especially during the pollination stage, high-cost pure pollen has high viscosity, poor dispersion, and low activity. Manual pollination of individual flowers results in excessive pollen spillage, severely hindering the production of male sterile tobacco lines and hybrids. Therefore, with the high cost of pollen collection, how to improve pollen utilization efficiency, increase pollen germination rate, and reduce pollen dosage and cost has become one of the key technologies in tobacco hybrid seed production and male sterile line breeding.

[0003] To address the above problems, researchers invented solid medium pollen technology, which reduces the amount of pollen required by mixing an inert solid medium with pure pollen and adds active ingredients to increase the pollen germination rate. In existing reports on solid medium pollen, inert media such as soluble starch have problems with inconsistent particle size and serious cohesion, which have led to uneven pollination in actual production and the problem of insignificant results has gradually become prominent. Active ingredients, such as the dried stamens and pistils of plant homologous plants reported in the literature, have problems with unclear active substances, difficult concentration control, and inability to be water-soluble and efficiently utilized. How to achieve uniform dispersion of pollen particles, clarify the active ingredients and their content, and maximize the promoting effect of active substances has become the development direction of better solid medium pollen. The application of nanomaterials provides a good idea for solving this problem. Preparing composite pollen with nanomaterials that have uniform particle size, hydrophobicity, high specific surface area, and the ability to efficiently load active substances is an effective measure.

[0004] In response to the above-mentioned problems, the present application proposes a method for preparing composite pollen based on carbon nanopowder. Summary of the Invention

[0005] The purpose of the present invention is to propose a method for preparing composite pollen based on carbon nanopowder. The main purpose is to propose a method for preparing composite pollen that can be evenly dispersed, save pollen usage, and improve pollen vitality. It aims to solve the problems existing in current crop, especially tobacco hybrid seed production, such as the difficulty in preparing solid medium pollen, large activity differences, and difficulty in dispersing active ingredients, which lead to the technical problem that the cost of hybrid seed production is difficult to effectively control.

[0006] In order to solve the above technical problems, the present invention adopts the following technical solutions:

[0007] A method for preparing composite pollen based on carbon nanopowder, the method comprising the following steps:

[0008] S1. Dissolving the active ingredient to prepare a solution of a specific concentration;

[0009] S2. Weigh carbon nanopowder and add the carbon nanopowder to the solution prepared in step S1 according to the proportion;

[0010] S3, stirring thoroughly until the carbon nanopowder and liquid are evenly mixed;

[0011] S4. Place the mixture in an oven with air blowing at 30°C to dry the liquid on the surface of the mixture, adjust the temperature to 40-50°C, dry, and aggregate the solid into blocks;

[0012] S5, grinding the aggregated carbon powder solid into powder in a mortar;

[0013] S6. The above active powder is mixed with pure pollen in a certain proportion and then sealed for storage to prepare high-activity composite pollen based on carbon nanopowder.

[0014] Furthermore, in step S1, the active substance is quercetin, the concentration of the prepared solution is 10 to 500 mg / L, and the liquid used to prepare the solution is anhydrous ethanol.

[0015] Furthermore, in step S2, the particle size of the carbon nanopowder is 20 nm, and the mass volume ratio of the carbon nanopowder to the quercetin solution is 1 g: 4-5 ml.

[0016] Furthermore, in step S3, the carbon nanopowder is mixed with the liquid to form a paste.

[0017] Furthermore, in step S4, the air-drying time is 2 to 3 hours, and the drying time is 3 to 4 hours.

[0018] Furthermore, in step S5, the carbon powder solid needs to be mortared until there are no obvious large aggregated particles.

[0019] Furthermore, in step S6, the mass ratio of the active medium powder to the pure pollen is 1 to 2:1.

[0020] Compared with the prior art, the present invention has at least the following beneficial effects:

[0021] 1. The present invention proposes a method for preparing composite pollen based on carbon nanopowder. After dissolving a specific concentration of the active ingredient quercetin in anhydrous ethanol, the mixture is then mixed with nano-scale high-specific surface area carbon powder in a liquid form. A specific mass of quercetin is dispersed and loaded on the carbon nanoparticles to the greatest extent possible, thereby enhancing the promoting effect of the water-insoluble quercetin on pollen, increasing the pollen germination rate, and reducing the amount of pollen used.

[0022] 2. The present invention proposes a method for preparing composite pollen based on carbon nanopowder. Commercially available carbon nanopowder is loaded with active substances and then mixed with pollen, especially tobacco pollen. The carbon powder is nano-scale particles with uniform particle size and high specific surface area. It can effectively adsorb quercetin particles dissolved in ethanol and then be mixed with pure pollen particles with larger particle size. It can be effectively dispersed around the pollen grains, thereby improving the germination rate of pollen, reducing the amount of pollen used, and ensuring the quality of pollination. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 The present invention is a schematic flow chart of a method for preparing composite pollen based on carbon nanopowder.

[0024] Figure 2 This is an in vitro germination diagram of pure pollen from control group 1 of the present invention.

[0025] Figure 3 This is the in vitro germination diagram of the zinc oxide nanocomposite pollen of the control group 2 of the present invention.

[0026] Figure 4 This is an in vitro germination diagram of Example 1 of the carbon nanopowder-based composite pollen of the present invention. DETAILED DESCRIPTION

[0027] like Figure 1-4 In order to make the purpose, technical solutions and advantages of the present invention more clearly understood, the present invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention.

[0028] The test materials used in the following examples were: MS Yunyan 87 tobacco male parent flowers with reddish tips and petals that had not yet opened or were in the initial opening stage. The pollen sacs were retained, and the remaining parts were removed. The flowers were air-dried until the sacs naturally opened. The pollen was sieved out and placed in a 50 ml PE tube, sealed, and stored at -80°C until use. The pollen was removed for in vitro germination experiments. All of the above pollen was produced by Yuxi Zhongyan Seed Co., Ltd.

[0029] Example 1

[0030] The present invention discloses a method for preparing composite pollen based on carbon nanopowder, specifically comprising the following steps: weighing quercetin and dissolving it in anhydrous ethanol to prepare a 10 mg / L aqueous solution, and measuring 15 ml of the solution into a 100 ml beaker for use. Separately weigh 4.5 g of commercially available 20 nm carbon powder and add it to the prepared quercetin ethanol solution, stirring, and continuing stirring until the solid and liquid are separated and the mixture becomes a viscous paste. The sample is then transferred to a 30°C oven and air-dried for 3 hours until the liquid disappears. The oven is then adjusted to 40°C and the dried solid is in a block-like shape. After stirring with a spatula, no significant viscosity is observed. The agglomerated solid is removed and ground into a mortar and pestle until no large particles are observed. Weigh 1 g of the quercetin-loaded nanocarbon powder and mix it with preserved MS Yunyan 87 pure pollen in a 1:1 mass ratio. The mixture is thoroughly mixed to prepare composite pollen for in vitro germination experiments.

[0031] A 10% sucrose aqueous solution was used as the liquid culture medium (without adding any active ingredients). 1 mL of the culture medium was pipetted onto the concave portion of a glass slide. Pure pollen was used as a control. Composite pollen was evenly sprinkled on the culture medium. The pollen and culture medium were mixed by pipetting three times. The above-mentioned glass slide was placed in a culture box and cultured in a dark constant temperature incubator at 28°C for 4 hours. The culture was then frozen at low temperature. After culture, the growth of pollen tubes was observed under an Olympus BX51 optical microscope. Three fields of view were randomly measured for each sample. The germination standard was that the pollen tube length exceeded the pollen length. The average value was taken to calculate the pollen germination rate.

[0032] Example 2

[0033] The present invention discloses a method for preparing composite pollen based on carbon nanopowder, specifically comprising the following steps: weighing quercetin and dissolving it in anhydrous ethanol to prepare a 50mg / L aqueous solution, and measuring 15ml into a 100ml beaker for use. Separately weigh 4.5g of commercially available 20nm carbon powder, add it to the prepared quercetin ethanol solution, stir, and continue stirring until the solid and liquid are separated and the mixture becomes a viscous paste. The sample is then transferred to a 30°C oven and air-dried for 3 hours until the liquid disappears. The oven is then adjusted to 40°C, and the dried solid is in a block-like shape. After stirring with a spatula, no significant viscosity is observed. The agglomerated solid is removed and ground into a mortar until no significant large particles are present. Weigh 1g of the nano-carbon powder loaded with quercetin and mix it with preserved MS Yunyan 87 pure pollen in a 1:1 mass ratio. The mixture is thoroughly mixed to prepare composite pollen for in vitro germination experiments.

[0034] A 10% sucrose aqueous solution was used as the liquid culture medium (without adding any active ingredients). 1 mL of the culture medium was pipetted onto the concave portion of a glass slide. Pure pollen was used as a control. Pure composite pollen was evenly sprinkled on the culture medium. The pollen and culture medium were mixed by pipetting three times. The above-mentioned glass slide was placed in a culture box and cultured in a dark constant temperature incubator at 28°C for 4 hours. The culture was then frozen at low temperature. After culture, the growth of the pollen tube was observed under an Olympus BX51 optical microscope. Three fields of view were randomly measured for each sample. The germination standard was that the pollen tube length exceeded the pollen length. The average value was taken to calculate the pollen germination rate.

[0035] Example 3

[0036] The present invention discloses a method for preparing composite pollen based on carbon nanopowder, specifically comprising the following steps: weighing quercetin and dissolving it in anhydrous ethanol to prepare a 200mg / L aqueous solution, and measuring 15ml into a 100ml beaker for use. Separately weigh 4.5g of commercially available 20nm carbon powder, add it to the prepared quercetin ethanol solution, stir, and continue stirring until the solid and liquid are not separated and become a sticky paste. The sample is then transferred to a 30°C oven and air-dried for 3h until the liquid disappears. The oven is adjusted to 40°C, and the dried solid is in a block shape. After stirring with a spatula, no obvious viscosity is observed. The agglomerated solid is removed and ground into a mortar until no obvious large particles are present. Weigh 1g of the nano-carbon powder loaded with quercetin and mix it with preserved MS Yunyan 87 pure pollen in a mass ratio of 1:1. The mixture is thoroughly mixed to prepare composite pollen for in vitro germination experiments.

[0037] A 10% sucrose aqueous solution was used as the liquid culture medium (without adding any active ingredients). 1 mL of the culture medium was pipetted onto the concave portion of a glass slide. Pure pollen was used as a control. Composite pollen was evenly sprinkled on the culture medium. The pollen and culture medium were mixed by pipetting three times. The above-mentioned glass slide was placed in a culture box and cultured in a dark constant temperature incubator at 28°C for 4 hours. The culture was then frozen at low temperature. After culture, the growth of pollen tubes was observed under an Olympus BX51 optical microscope. Three fields of view were randomly measured for each sample. The germination standard was that the pollen tube length exceeded the pollen length. The average value was taken to calculate the pollen germination rate.

[0038] Example 4

[0039] The present invention discloses a method for preparing composite pollen based on carbon nanopowder, specifically comprising the following steps: weighing quercetin and dissolving it in anhydrous ethanol to prepare a 500mg / L aqueous solution, and measuring 15ml into a 100ml beaker for use. Separately weigh 4.5g of commercially available 20nm carbon powder, add it to the prepared quercetin ethanol solution, stir, and continue stirring until the solid and liquid are not separated and become a sticky paste. The sample is then transferred to a 30°C oven and air-dried for 3h until the liquid disappears. The oven is adjusted to 40°C, and the dried solid is in a block shape. After stirring with a spatula, no obvious viscosity is observed. The agglomerated solid is removed and ground into a mortar until no obvious large particles are present. Weigh 1g of the nano-carbon powder loaded with quercetin and mix it with preserved MS Yunyan 87 pure pollen in a mass ratio of 1:1. The mixture is thoroughly mixed to prepare composite pollen for in vitro germination experiments.

[0040] A 10% sucrose aqueous solution was used as the liquid culture medium (without adding any active ingredients). 1 mL of the culture medium was pipetted onto the concave portion of a glass slide. Pure pollen was used as a control. Composite pollen was evenly sprinkled on the culture medium. The pollen and culture medium were mixed by pipetting three times. The above-mentioned glass slide was placed in a culture box and cultured in a dark constant temperature incubator at 28°C for 4 hours. The culture was then frozen at low temperature. After culture, the growth of pollen tubes was observed under an Olympus BX51 optical microscope. Three fields of view were randomly measured for each sample. The germination standard was that the pollen tube length exceeded the pollen length. The average value was taken to calculate the pollen germination rate.

[0041] Control group 1

[0042] The difference between the control group 1 and Example 1 is that the control group 1 uses pure pollen.

[0043] Control group 2

[0044] The difference between the control group 2 and Examples 1 to 4 is that the control group 2 uses 10 mg / -500 mg / L of 15 nm zinc oxide particles to prepare nanocomposite pollen.

[0045] The statistical results are shown in Table 1:

[0046] Table 1 Statistics of in vitro germination rates of different composite pollen

[0047]

[0048]

[0049] As can be seen from Table 1, compared with the pure pollen of the control group 1, after the nano-carbon powder loaded with different concentrations of quercetin was mixed with the pure pollen in a 1:1 ratio, the germination rate of the composite pollen of 10 mg / L to 500 mg / L was significantly improved relative to the pure pollen, which was increased by 10.69% to 12.28%, indicating that the nano-carbon powder loaded with quercetin can effectively improve the pollen germination rate. As can be seen from the accompanying drawings, at the same germination time, the pollen tube length of the composite pollen of the nano-carbon powder in the present invention is much longer than that of the pure pollen, which will inevitably improve the pollination efficiency, and the 1:1 ratio can save 50% of the pollen dosage and reduce the seed production cost, indicating that the innovative achievements of the present invention are significant.

[0050] Compared with the zinc oxide nanoparticles in the control group 2, the zinc oxide nanoparticles loaded with different concentrations of quercetin and composite pollen did not promote pollen germination. Instead, it reduced the pollen germination rate and had a certain inhibitory effect on pollen germination. This also shows the advantage of the nano-carbon powder composite pollen in the present invention. The reason for the difference in results may be due to the different specific surface areas of the nanoparticles.

[0051] Finally, it should be noted that the above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art will be able to modify the technical solutions described in the aforementioned embodiments or substitute equivalents for some of the technical features. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention shall be included within the scope of protection of the present invention.

Claims

1. A method for preparing composite pollen based on carbon nanopowder, characterized in that: The method comprises the following steps: S1. Dissolving the active ingredient, wherein the active ingredient is quercetin, and the concentration of the prepared solution is 10-500 mg / L, and the liquid used for preparing the solution is anhydrous ethanol; S2. Weigh carbon nanopowder, the particle size of the carbon nanopowder is 20 nm, the mass volume ratio of the carbon nanopowder to the quercetin solution is 1 g: 4-5 ml, and add the carbon nanopowder to the solution prepared in step S1 according to the ratio; S3, stirring thoroughly until the carbon nanopowder and liquid are evenly mixed; S4, placing the above mixture in an oven with air blowing at 30°C, air-drying the liquid on the surface of the mixture for 2 to 3 hours, adjusting the temperature to 40 to 50°C, drying for 3 to 4 hours, and agglomerating the solid into blocks; S5, grinding the aggregated carbon powder solid into powder in a mortar; S6. The active medium powder and the pure pollen are fully mixed in a ratio of 1 to 2:1, and the mixture is sealed and stored to prepare a high-vitality composite pollen based on carbon nanopowder. Pure pollen is tobacco pollen.

2. The method for preparing composite pollen based on carbon nanopowder according to claim 1, characterized in that: In step S3, the carbon nanopowder is mixed with the liquid to form a paste.

3. The method for preparing composite pollen based on carbon nanopowder according to claim 1, characterized in that: In step S5, the carbon powder solid needs to be ground into a mortar until there are no obvious large aggregated particles.

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