Use of sulfur nanoparticles in promoting differentiation and regeneration of plant callus

Abstract

The application provides application of sulfur nanoparticles in promoting differentiation and regeneration of plant callus. The application research finds that after treatment of plant proembryo with the sulfur nanoparticles, the number of regenerated sprouts on the induced plant callus is significantly increased compared with a control group, indicating that the sulfur nanoparticles can significantly improve the differentiation and regeneration capacity of the plant callus. The application provides a new method for solving the difficulty in differentiation and regeneration of the plant callus, and has wide application prospect in genetic improvement of crops.

Description

Technical Field

[0001] This invention relates to the technical field of plant molecular biology, specifically to the application of sulfur nanoparticles in promoting the differentiation and regeneration of plant callus tissue. Background Technology

[0002] Maize and wheat are among the most widely planted and highest-yielding food crops globally, serving as core carriers for ensuring food security and supporting sustainable agricultural development. Their genetic improvement and the breeding of superior varieties have always been key research areas in agriculture. Callus differentiation and regeneration technology, as a crucial technology for crop asexual reproduction, variety improvement, and germplasm resource innovation, plays an irreplaceable role in breeding practice, enabling the rapid fixation and large-scale propagation of superior traits. However, as typical monocotyledonous plants, the callus differentiation and regeneration process in maize and wheat has long been constrained by multiple technical bottlenecks, resulting in low application efficiency and severely restricting the breeding progress and promotion speed of superior maize and wheat varieties.

[0003] Currently, the core shortcomings of callus differentiation and regeneration in maize and wheat are mainly reflected in two aspects: First, the differentiation and regeneration efficiency is generally low. Even if callus formation is successfully induced, it is difficult to efficiently differentiate into complete plants. Most callus tissues suffer from differentiation arrest and abnormal development, significantly increasing breeding costs and cycles. Second, it is highly dependent on specific genotypes. Most maize and wheat varieties (lines) with important agronomical value (such as stress resistance, high yield, and high quality) cannot be induced to form differentiated callus tissue due to their own genotype limitations, or their differentiation and regeneration efficiency is extremely low. This results in a large number of excellent germplasm resources not being able to be efficiently utilized through callus regeneration technology. These shortcomings together prevent maize and wheat callus differentiation and regeneration technology from realizing its due value, becoming a major bottleneck restricting the genetic improvement of these two major food crops.

[0004] In recent years, nanomaterials, with their unique physicochemical properties such as small particle size, large specific surface area, easy surface functionalization, good biocompatibility, and easy penetration of plant cell barriers, have been widely used in biomedicine, environmental science, and other fields, while also providing new ideas and breakthroughs for technological innovation in agriculture. In plant science research, numerous studies have reported the application of nanomaterials in plant growth regulation, nutrient absorption promotion, and stress resistance enhancement. Some studies have used nanomaterials as novel delivery carriers, successfully achieving efficient and safe delivery of functional proteins and small molecule regulatory substances into plant cells, effectively overcoming the limitations of traditional technologies in material delivery and demonstrating broad application potential. However, it is worth noting that current research on the application of nanomaterials in the plant field is mostly focused on plant growth regulation and nutrient utilization; their application in callus differentiation and regeneration in crops is still rarely reported. Summary of the Invention

[0005] To overcome the aforementioned defects and shortcomings in the existing technology, this invention provides the application of sulfur nanoparticles in promoting the differentiation and regeneration of plant callus tissue.

[0006] The first objective of this invention is to provide the application of sulfur nanoparticles in promoting the differentiation and regeneration of plant callus tissue.

[0007] The second objective of this invention is to provide a method for promoting the differentiation and regeneration of plant callus tissue.

[0008] This invention claims protection for the following:

[0009] The application of sulfur nanoparticles in promoting the differentiation and regeneration of plant callus tissue, wherein the sulfur nanoparticles are sulfur nanoparticles with surface modified with polyethyleneimine.

[0010] Preferably, the plant includes a monocotyledonous plant.

[0011] More preferably, the monocotyledonous plant is maize.

[0012] More preferably, the monocotyledonous plant is wheat.

[0013] Preferably, the method for preparing the sulfur nanoparticles includes the following steps:

[0014] S1. A hydrothermal reaction was carried out with sulfur powder, polyethylene glycol and sodium hydroxide to obtain an SNPs-PEG solution;

[0015] S2. Reaction of polyethyleneimine and SNPs-PEG solution, separation using a membrane with a molecular weight cutoff of 2000 Da, collection of the retentate yields sulfur nanoparticles.

[0016] More preferably, in step S1, the ratio of sulfur powder, polyethylene glycol and sodium hydroxide is (0.175~0.525) g: (0.375~1.125) mL: (0.5~1.5) g.

[0017] More preferably, the ratio of sulfur powder, polyethylene glycol and sodium hydroxide is 0.35 g: 0.75 mL: 1 g.

[0018] More preferably, the polyethylene glycol has a molecular weight of 400.

[0019] More preferably, the sulfur powder is sublimated sulfur powder.

[0020] More preferably, in step S1, the hydrothermal reaction is carried out at 160–180°C for 1–3 h.

[0021] More preferably, the hydrothermal reaction is carried out at 170°C for 2 hours.

[0022] More preferably, in step S2, the volume ratio of the polyethyleneimine to the SNPs-PEG solution is 1:(3-5).

[0023] More preferably, the volume ratio of the polyethyleneimine to the SNPs-PEG solution is 1:4.

[0024] More preferably, in step S2, the reaction conditions are 65-75°C for 3-5 hours.

[0025] More preferably, the reaction conditions are 70°C for 4 hours.

[0026] A method for promoting plant callus differentiation and regeneration involves treating plant embryos with sulfur nanoparticles.

[0027] The sulfur nanoparticles are sulfur nanoparticles with a surface modified with polyethyleneimine.

[0028] Preferably, the plant includes a monocotyledonous plant.

[0029] More preferably, the monocotyledonous plant is maize.

[0030] More preferably, the monocotyledonous plant is wheat.

[0031] Preferably, the method for preparing the sulfur nanoparticles includes the following steps:

[0032] S1. Sulfur powder, polyethylene glycol and sodium hydroxide undergo a hydrothermal reaction to obtain an SNPs-PEG solution;

[0033] S2. Reaction of polyethyleneimine and SNPs-PEG solution, separation using a membrane with a molecular weight cutoff of 2000 Da, collection of the retentate yields sulfur nanoparticles.

[0034] More preferably, in step S1, the ratio of sulfur powder, polyethylene glycol and sodium hydroxide is (0.175~0.525) g: (0.375~1.125) mL: (0.5~1.5) g.

[0035] More preferably, the ratio of sulfur powder, polyethylene glycol and sodium hydroxide is 0.35 g: 0.75 mL: 1 g.

[0036] More preferably, the polyethylene glycol has a molecular weight of 400.

[0037] More preferably, the sulfur powder is sublimated sulfur powder.

[0038] More preferably, in step S1, the hydrothermal reaction is carried out at 160–180°C for 1–3 h.

[0039] More preferably, the hydrothermal reaction is carried out at 170°C for 2 hours.

[0040] More preferably, in step S2, the volume ratio of the polyethyleneimine to the SNPs-PEG solution is 1:(3-5).

[0041] More preferably, the volume ratio of the polyethyleneimine to the SNPs-PEG solution is 1:4.

[0042] More preferably, in step S2, the reaction conditions are 65-75°C for 3-5 hours.

[0043] More preferably, the reaction conditions are 70°C for 4 hours.

[0044] Preferably, the concentration of the sulfur nanoparticles is 125–400 mg / L.

[0045] Preferably, the treatment time for the sulfur nanoparticles is 5 to 10 minutes.

[0046] Compared with the prior art, the present invention has the following beneficial effects:

[0047] This invention provides the application of sulfur nanoparticles in promoting the differentiation and regeneration of plant callus. The study found that treatment of plant embryos with sulfur nanoparticles significantly increased the number of regenerated shoots on the induced callus compared to the control group, indicating that sulfur nanoparticles can significantly improve the differentiation and regeneration capacity of plant callus. This invention provides a new method for solving the difficulties in plant callus differentiation and regeneration, and has broad application prospects in crop genetic improvement. Attached Figure Description

[0048] Figure 1 Preparation and characterization of sulfur nanoparticles; a: Schematic diagram of the synthesis steps of sulfur nanoparticles; b: TEM image of sulfur nanoparticles; c: FT-IR spectrum of sulfur nanoparticles; d: Fluorescence emission spectra of sulfur nanoparticles at different excitation wavelengths; e: Reaction of sulfur nanoparticles to hydrogen peroxide (H2O2), hydroxyl radicals (·OH), and superoxide anions (O2·) - The clearance rate of ).

[0049] Figure 2 The effect of sulfur nanoparticles on the differentiation and regeneration of maize callus tissue.

[0050] Figure 3 The effect of sulfur nanoparticles on the differentiation and regeneration of wheat callus tissue. Detailed Implementation

[0051] The present invention will be further illustrated below with reference to specific embodiments, but the embodiments do not limit the present invention in any way. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in this technical field.

[0052] Unless otherwise specified, all reagents and materials used in the following examples are commercially available.

[0053] Example 1: Preparation and characterization of sulfur nanoparticles (SNPs)

[0054] I. Experimental Methods

[0055] 1. Synthesis of SNPs

[0056] The synthesis of SNPs is carried out according to the following steps ( Figure 1 a):

[0057] (1) Weigh 0.28 g of sublimed sulfur powder, 0.6 mL of PEG (molecular weight of 400 Da) and 0.8 g of NaOH, add 10 mL of deionized water, react at 170 °C for 2 h, cool to room temperature to obtain SNPs-PEG solution, and irradiate the solution with ultraviolet light to show blue fluorescence;

[0058] (2) Mix 0.4 mL PEI (molecular weight of 1800 Da) and 1.6 mL SNPs-PEG solution, stir magnetically at 70℃ for 4 h, then dialyze with a 2000 Da dialysis bag for 12 h, take the retentate to obtain SNPs solution, and irradiate the solution with a UV lamp to show green fluorescence.

[0059] 2. Characterization of SNPs

[0060] SNPs were characterized using transmission electron microscopy (TEM), fluorescence spectroscopy, and Fourier transform infrared spectroscopy, respectively. Kits were used to detect the SNPs' effects on hydrogen peroxide (H₂O₂), hydroxyl radicals (·OH), and superoxide anions (O₂··OH). - The clearance rate of ).

[0061] II. Experimental Results

[0062] TEM images of SNPs, such as Figure 1 As shown in b, SNPs are uniformly dispersed in aqueous solution with an average particle size of 1.5 nm and a lattice interstices of 0.23 nm.

[0063] FT-IR spectroscopy shows ( Figure 1 (c) The sample exhibits a distinct NH characteristic absorption peak, located at 3352 cm⁻¹. -1 (NH stretching vibration) and 1582 cm-1 (NH bending vibration); simultaneously at 2950 cm -1 With 2820 cm -1 An absorption peak for the CH stretching vibration of -CH2- appears at 1450 cm⁻¹, and at 1450 cm⁻¹. -1 A significant peak was observed at 1112 cm, corresponding to the in-plane bending vibration of CH. -1 The absorption peak at 1002 cm⁻¹ can be attributed to the CN stretching vibration, indicating that PEI was successfully capped on the surface of SNPs-PEG. -1 The new absorption peak at 775 cm⁻¹ is attributed to SNS bonding. -1 The location may be attributed to SN stretching, 660 cm -1 This is attributed to the SO stretching vibration. The presence of the SN bond indicates a chemical reaction between sulfur and the amino group on the PEI, demonstrating the successful synthesis of SNPs.

[0064] Figure 1 In the figure, d represents the fluorescence emission spectrum of SNPs at different excitation wavelengths, showing that the prepared SNPs are wavelength-dependent.

[0065] SNPs inhibit hydrogen peroxide (H2O2), hydroxyl radicals (·OH), and superoxide anions (O2·). - The clearance rates were 15.94±0.93%, 35.18±3.23%, and 66.71±1.09%, respectively. Figure 1 The result (e) indicates that SNPs possess the ability to scavenge reactive oxygen species (ROS) in vitro, directly reacting with ROS to reduce their concentration. These results demonstrate that SNPs can directly mimic enzyme activity in vitro to scavenge ROS, and can function as nano-enzymes to perform ROS scavenging.

[0066] Example 2: Effects of sulfur nanoparticles on the differentiation and regeneration of maize callus tissue

[0067] I. Experimental Methods

[0068] 1. Treatment of immature embryos

[0069] Select maize ears (variety: Woyu No. 3, KN5585) 10 days after pollination, disinfect with 75% ethanol solution (v / v) for 10 min, and rinse 3 times with sterile distilled water. Place in a laminar flow hood and remove 1 / 3 of the pericarp, pick out immature embryos of about 1.5 mm, and treat them in a culture medium containing 400 mg / L SNPs solution prepared in Example 1 for 5 min.

[0070] 2. Culture of callus tissue

[0071] After treatment, the embryos were blotted dry with sterile filter paper, transferred to 1 / 2 MS medium, shield side up, and co-cultured at 18°C ​​in the dark for 72 h.

[0072] 3. Regeneration of callus tissue

[0073] After culture, the immature embryos were transferred to 1 / 2 MS medium and cultured in the dark at 25°C for 28 days. Healthy callus tissue was selected and transferred to differentiation medium (MS + 6-BA 2 mg / L + NAA 0.2 mg / L) and cultured at 26°C with 16 h light / 8 h dark for 5 days. Differentiated green shoots were transferred to regeneration medium (1 / 2 MS + IBA 0.5 mg / L) and cultured at 26°C with 16 h light for 14 days. After rooting, they were transplanted into nutrient soil and cultured at 26±1°C, 70% humidity, and 200 μmol / L. m -2 s -1 Corn plants were obtained by culturing under PAR light conditions for 30 days.

[0074] II. Experimental Results

[0075] The number of regenerated shoots on the callus in the SNPs treatment group was 11.33±1.47, while the number of regenerated shoots on the callus in the control group was 5±0.71. The number of regenerated shoots in the SNPs treatment group was 1.26 times higher than that in the control group, indicating that the SNPs prepared in Example 1 significantly improved the differentiation and regeneration capacity of maize callus.

[0076] Example 3: Effects of sulfur nanoparticles on wheat callus differentiation and regeneration

[0077] I. Experimental Methods

[0078] 1. Pretreatment of immature embryos

[0079] Wheat ears (varieties: Triticum aestivum L. cv. Fielder, Bobwhite) 12 days after pollination were selected. After removing the husks, they were sterilized with 75% ethanol solution (v / v) for 2 min, followed by treatment with 2.5% sodium hypochlorite solution (containing 0.02% Tween-20, v / v) for 10 min, and rinsed three times with sterile distilled water. Immature embryos (approximately 1.0 mm in size, with visible shield but not yet fully differentiated) were extracted in a laminar flow hood. The embryos were then placed in a culture medium containing 125 mg / L SNPs solution prepared in Example 1 for 10 min.

[0080] 2. Culture of callus tissue

[0081] Remove the treated embryos, blot dry the surface liquid with sterile filter paper, and transfer them to co-culture medium (MS + 2,4-D 2.0 mg / L + 100 μM AS) with the shield side facing up. Co-culture at 22℃ in the dark for 48 h.

[0082] 3. Regeneration of callus tissue

[0083] After callus culture, immature embryos were transferred to 1 / 2 MS medium and cultured in the dark for 28 days. Healthy callus tissue was selected and transferred to differentiation medium (MS + 6-BA 1.5 mg / L + IAA 0.2 mg / L), and cultured at 26℃ under 16 h light / 8 h dark conditions for 10 days. The resulting green shoots were transferred to rooting medium (1 / 2 MS + IBA 0.5 mg / L) and cultured at 26℃ under 16 h light conditions for 14 days. After the root system was fully developed, the regenerated plants were transplanted into nutrient soil and cultured at 26±1℃, 70% relative humidity, and 200 μmol / L. m -2 s -1 Wheat plants were obtained by culturing under PAR light conditions for 30 days.

[0084] II. Experimental Results

[0085] The number of regenerated shoots on wheat callus in the SNPs treatment group was 10.88±1.68, while the number of regenerated shoots on callus in the control group was 2.33±0.88. The number of regenerated shoots in the SNPs treatment group was 3.67 times higher than that in the control group, indicating that the SNPs prepared in Example 1 significantly improved the differentiation and regeneration capacity of wheat callus.

[0086] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. The application of sulfur nanoparticles in promoting plant callus differentiation and regeneration, characterized in that, The sulfur nanoparticles are sulfur nanoparticles with a surface modified with polyethyleneimine. The plants include monocotyledonous plants; The monocotyledonous plant is corn or wheat; The method for preparing the sulfur nanoparticles includes the following steps: S1. A hydrothermal reaction was carried out with sulfur powder, polyethylene glycol and sodium hydroxide to obtain an SNPs-PEG solution; S2. Reaction of polyethyleneimine and SNPs-PEG solution, separation using a membrane with a molecular weight cutoff of 2000 Da, collection of the retentate yields sulfur nanoparticles.

2. The application according to claim 1, characterized in that, In step S1, the ratio of sulfur powder, polyethylene glycol and sodium hydroxide is (0.175~0.525) g : (0.375~1.125) mL : (0.5~1.5) g.

3. The application according to claim 2, characterized in that, In step S2, the volume ratio of the polyethyleneimine to the SNPs-PEG solution is 1:(3-5).

4. A method for promoting the differentiation and regeneration of plant callus tissue, characterized in that, Plant embryos were treated with sulfur nanoparticles. The sulfur nanoparticles are sulfur nanoparticles with a surface modified with polyethyleneimine. The plants include monocotyledonous plants; The monocotyledonous plant is corn or wheat; The method for preparing the sulfur nanoparticles includes the following steps: S1. A hydrothermal reaction was carried out with sulfur powder, polyethylene glycol and sodium hydroxide to obtain an SNPs-PEG solution; S2. Reaction of polyethyleneimine and SNPs-PEG solution, separation using a membrane with a molecular weight cutoff of 2000 Da, collection of the retentate yields sulfur nanoparticles.

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