Use of a phenolic foam-nanomontmorillonite composition for the preparation of porous substrate blocks for rooting of somatic embryos

Abstract

The application belongs to the field of planting, and aims at the problem of single function of the existing plant rooting culture support, and specifically provides application of a phenolic foam-nano montmorillonite composition in preparation of a porous matrix block for rooting of somatic embryo seedlings. Any one of the phenolic foam-nano montmorillonite compositions Y1-Y3 is disclosed to be applied in preparation of a porous matrix block for rooting of somatic embryo seedlings: Y1 is applied in preparation of a porous matrix block for rooting of somatic embryo seedlings; Y2 is applied in promotion of plant lateral root formation; and Y3 is applied in plant planting. The composition is obtained by foaming and solidification of the phenolic foam-nano montmorillonite composition after adding nano montmorillonite into a phenolic resin prepolymer. The matrix block prepared from the phenolic foam-nano montmorillonite composition can significantly promote lateral root formation of plants, especially Picea somatic embryo seedlings, greatly enhance the absorption and fixation capacity of the seedlings, and significantly improve the growth amount of seedling height, the lateral root occurrence proportion and the average lateral root number.

Description

Technical Field

[0001] This invention belongs to the field of planting, specifically relating to the application of a phenolic foam-nanomontmorillonite composition in the preparation of porous substrate blocks for the rooting of seedlings. Background Technology

[0002] Somatic embryogenesis is a key technology for the large-scale propagation of superior conifer varieties such as spruce. However, the rooting stage of somatic embryo seedlings still faces technical bottlenecks such as low rooting rate, poor root structure (sparse lateral roots), and low transplant survival rate.

[0003] Currently used agar media have poor air permeability and high mechanical resistance, which easily leads to poor root development. While some studies have attempted to use various porous materials (such as rock wool and polyurethane foam) as support to replace agar and improve aeration, these materials primarily address oxygen supply and have limited physiological regulation of root development, especially in inducing lateral root growth. The number and distribution of lateral roots are key factors determining the efficiency of water and nutrient absorption in seedlings and the success of transplanting. Therefore, developing a novel intelligent substrate that not only provides good physical support but also actively regulates root development (especially promoting lateral root growth) is crucial for improving the quality of spruce seedlings. Summary of the Invention

[0004] To address the limitations of existing rooting culture supports with limited functionality, this invention provides applications of phenolic foam-nanomontmorillonite compositions, wherein the application is any one of Y1-Y3: Application of Y1 in the preparation of porous substrate blocks for rooting of somatic embryos; Application of Y2 in promoting lateral root formation in plants; Application of Y3 in plant cultivation; The composition is made from raw materials including phenolic resin prepolymer and nano-montmorillonite, wherein the mass ratio of the phenolic resin prepolymer to nano-montmorillonite is 18950:360-900.

[0005] In the above applications, the phenolic resin prepolymer is a mixture composed of phenolic compounds, aldehyde compounds, and additives, serving as a precursor for preparing phenolic foaming resin. The phenolic compounds are phenol (CAS No.: 108-95-2); the aldehyde compounds are formaldehyde (liquid, CAS No.: 8013-13-6) and paraformaldehyde (CAS No.: 30525-89-4); and the additives are urea (CAS No.: 57-13-6), polyethylene dicocarbamate (PEG 400 DILAURATE, CAS No.: 68139-91-3), and water. In one embodiment of this application, the phenolic resin prepolymer is a mixture obtained by mixing phenol:formaldehyde:paraformaldehyde:urea:polycocarbamate:water in a molar ratio of 20:6:12:1:3:1.

[0006] In some embodiments, the nano-montmorillonite (CB No. CB818992933) refers to montmorillonite particles with a particle size (diameter) of 50-150 nm.

[0007] In the above applications, the composition is a phenolic foam-nanomontmorillonite composition, which is a composition with phenolic foam as the substrate and nanomontmorillonite loaded with nanomontmorillonite obtained by adding nanomontmorillonite to a phenolic resin prepolymer, followed by foaming and curing. It can be used as a porous substrate for cultivating plants.

[0008] In the above applications, the composition not only provides an excellent physical and aeration environment, but also significantly promotes the formation and development of lateral roots in seedlings through its unique chemical properties, thereby cultivating high-quality seedlings with a well-developed root system.

[0009] In the above applications, the composition has an open porosity >95% and a pore size (diameter) range of 150-250 μm.

[0010] The porosity is the proportion of the volume occupied by open pores (i.e., holes that communicate with the outside world).

[0011] In the above applications, the foaming and curing process uses foaming agents, foam leveling agents, and curing agents as additives: The foaming agent can be obtained by mixing n-pentane (CAS No.: 109-66-0) and n-hexane (CAS No.: 110-54-3) in a weight ratio of 2:1. In one specific embodiment, the amount of foaming agent added satisfies the following condition: 3000 parts by weight of the foaming agent are added to 18950 parts by weight of the phenolic resin prepolymer.

[0012] The foam stabilizer is silicone oil (CAS No.: 63148-62-9). In one specific embodiment, the amount of foam stabilizer added satisfies the following condition: 500 parts by weight of silicone oil are added to 18950 parts by weight of phenolic resin prepolymer.

[0013] The curing agent is obtained by mixing p-toluenesulfonic acid (CAS No.: 104-15-4), phenolsulfonic acid (CAS No.: 1857-03-0), and phosphoric acid (CAS No.: 7664-38-2) in a weight ratio of 6:3:1. In one specific embodiment, the amount of curing agent added satisfies the following condition: 4000 parts by weight of the curing agent are added to 18950 parts by weight of the phenolic resin prepolymer.

[0014] In the above applications, the plant can be a woody plant, preferably a forest tree, specifically a pine family plant; the pine family plant can be a spruce genus plant, specifically European spruce (Picea spp.). Picea abies (L.) H. Karst.).

[0015] The present invention also provides the above-described phenolic foam-nanomontmorillonite composition.

[0016] The present invention also provides a method for preparing the above-mentioned phenolic foam-nano montmorillonite composition, the method comprising the following steps: A. Premixing: The nano-montmorillonite is dispersed in the phenolic resin prepolymer to form a suspension; B. Foaming and curing: The foaming agent, the foam leveling agent and the curing agent are added to the suspension, and then foaming and curing are performed to obtain the composition.

[0017] In the above method, the foaming and curing are carried out at 57.8°C.

[0018] The present invention also provides a method for preparing a cultivation substrate block, the substrate block being composed of the phenolic foam-nanomontmorillonite composition, the preparation method comprising the following steps: The composition was prepared according to the above-described method for preparing phenolic foam-nanomontmorillonite composition, and the composition was then cut, washed, and sterilized to obtain the matrix block.

[0019] In the above method, the cutting refers to cutting into the required shape and size, such as small pieces, wedges, etc.

[0020] The present invention also provides a method for promoting rooting of spruce spore seedlings, comprising the step of inoculating the spruce spore seedlings onto the substrate block of an adsorbed liquid rooting medium for cultivation.

[0021] In the above method, the spruce somatic embryo seedlings are obtained through somatic embryogenesis, specifically, the preferred somatic embryo seedlings are obtained by drying treatment of mature somatic embryos obtained from the differentiation of spruce embryogenic cell lines.

[0022] In the above method, during the cultivation process, the bottom of the substrate block is immersed in the liquid rooting medium, and the liquid rooting medium does not submerge the substrate block.

[0023] In the above method, the liquid rooting medium is a conventional liquid rooting medium already available in the prior art, and 1 / 2 LM liquid rooting medium can be used, with the specific formula as follows: 825 mg·L⁻¹ -1 NH4NO3, 950 mg·L -1 KNO3, 451.7 mg·L -1 MgSO4, 15.5 mg·L -1 H2BO3, 0.25 mg·L -1 CuSO4·5H2O, 10.5 mg·L -1 MnSO4·H2O, 2.08 mg·L -1 KI, 11 mg·L -1 CaCl2·2H2O, 170 mg·L -1 KH2PO4, 1.13 mg·L -1 Na2SO4·10H2O, 0.085 mg·L -1 CoCl2·8H2O, 18 mg·L -1 EDTANa2, 15 mg·L -1 FeSO4, 21.5 mg·L -1 ZnSO4·7H2O, 100 mg·L -1 Inositol, 0.5 mg·L -1 Niacin, 10.1 mg·L -1 Vitamin B1, 0.1 mg / L -1 Vitamin B6, 8 g·L -1 Sucrose, 4 g·L -1 Plant gel and 5 mg·L -1 Glutamine.

[0024] The above-mentioned method for promoting rooting of spruce seedlings also includes the step of transplanting the rooted spruce seedlings together with the substrate block.

[0025] The beneficial effects of this invention are as follows: 1. Significantly promotes lateral root development: The core innovation of this invention is the introduction of nano-montmorillonite into phenolic foam. Nano-montmorillonite possesses excellent ion exchange capacity and adsorption properties, possibly through the following synergistic mechanisms: Slow-release nutrition: Adsorbs macro- and micro-elements in the culture medium and slowly releases them around the roots, forming a local microenvironment conducive to the induction of root primordia and the development of lateral roots.

[0026] Improving the rhizosphere microecology: Providing habitats for beneficial microorganisms may promote symbiosis and indirectly stimulate root development.

[0027] Physical stimulation: Nanoparticles may provide mild physical stimulation to root tip cells, activating signaling pathways related to lateral root development.

[0028] 2. Dual Advantages Structure: Phenolic foam itself has the characteristics of moderate rigidity, uniform pores, good hydrophilicity, and resistance to high-pressure sterilization, providing ideal growth space and aeration conditions for roots. Its stability is superior to some flexible foams.

[0029] 3. Synergistic effect: The physical support of phenolic foam combined with the chemical regulation function of nano-montmorillonite produces a synergistic effect of "1+1>2", which not only improves the rooting rate and root system structure, transforming the root system from "tap root type" to "fibrous root type", but also greatly enhances the absorption and fixation capacity of seedlings.

[0030] 4. Improve transplant survival rate: The well-developed lateral root system enables seedlings to adapt to the post-transplant environment more quickly, significantly improving the transplant survival rate and early growth rate. Attached Figure Description

[0031] Figure 1 This is a schematic diagram of the microstructure and a statistical analysis of the pore size of the phenolic foam-nanomontmorillonite composition in Example 1 of the present invention. Figure 1 A is the SEM image of T1. Figure 1 B is a T5 SEM image. Figure 1 C is the SEM image of T6. Figure 1 D represents the statistical result of the number of pores of different sizes in T1. Figure 1 E represents the statistical results of the number of pores of different sizes in T5. Figure 1 F represents the statistical results of the number of different aperture sizes of T6.

[0032] Figure 2 This is a comparison of the root systems of spruce seedlings cultured in different substrates in Example 1 of the present invention. Figure 2 A represents the root phenotype diagram. Figure 2 B represents plant height. Figure 2 C represents the proportion of lateral root occurrence. Figure 2D represents the number of lateral roots. In the figure, * indicates a significance analysis result of P < 0.05, and ** indicates a significance analysis result of P < 0.01. Detailed Implementation

[0033] The present invention will now be described in further detail with reference to specific embodiments. The given embodiments are merely illustrative of the invention and not intended to limit its scope. The embodiments provided below can serve as a guide for further improvements by those skilled in the art and do not constitute a limitation on the invention in any way.

[0034] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available. All quantitative experiments in the following examples were performed in triplicate, and the results were averaged.

[0035] Example 1 The spruce seedlings were obtained from European spruce ( Picea abies The somatic embryogenesis method used by (L.) H. Karst. specifically involves obtaining optimized somatic embryos from mature somatic embryos differentiated from spruce embryogenic cell lines through drying treatment. For detailed steps, refer to "Optimized cryopreservation of embryogenic tissue of (L.) H. Karst." Picea abies based on differential scanning calorimetry" (https: / / doi.org / 10.1016 / j.cryobiol.2025.105278) and "Transcriptome and metabolome analyzes revealed that partial desiccation treatment promotes somatic embryo germination of Picea asperata through cell wall remodeling mediated by transcription factorPaMYB12" (https: / / doi.org / 10.1016 / j.indcrop.2025.121542).

[0036] 1. Matrix preparation This embodiment sets up three types of matrix blocks: matrix blocks T5 and T6 prepared with two different phenolic foam-nanomontmorillonite compositions, a control matrix block T1 prepared with phenolic foam without added nanomontmorillonite, and a matrix block prepared with 4 g·L⁻¹. -1 The control substrate block CK was prepared using agar solid culture medium.

[0037] The preparation methods for the three types of matrix blocks in this embodiment are as follows: 1.1 Preparation of matrix blocks T5 and T6 using phenolic foam-nanomontmorillonite composition Nano-montmorillonite (CB No. CB818992933) refers to montmorillonite particles with a particle size (diameter) of 50-150 nm.

[0038] The phenolic resin prepolymer in this embodiment is a precursor to phenolic foaming resin, obtained by mixing phenol, formaldehyde, paraformaldehyde, urea, polyethylene glycol dicosinate, and water in a molar ratio of 20:6:12:1:3:1. The preparation methods for matrix blocks T5 and T6 are as follows: A' Premixing: The nano-montmorillonite is dispersed in the phenolic resin prepolymer to form a suspension; specifically: Take 18.95 kg of phenolic resin prepolymer, and add 360 g and 900 g of nano-montmorillonite respectively. Stir at high speed to disperse it evenly, forming stable suspension 1 (360 g of nano-montmorillonite added) and suspension 2 (900 g of nano-montmorillonite added) respectively.

[0039] B' Foaming and curing: The foaming agent, foam leveling agent and curing agent are added to the suspension, and the suspension is foamed and cured at high temperature to obtain the phenolic foam-nanomontmorillonite composition; Specifically: The foaming agent is a mixture of n-pentane and n-hexane in a mass ratio of 2:1. Specifically, the amount of n-pentane used is 2.0 kg, and the amount of n-hexane used is 1.0 kg.

[0040] The foam stabilizer is silicone oil, and the amount added is 0.5 kg.

[0041] The curing agent is obtained by mixing p-toluenesulfonic acid, phenolsulfonic acid and phosphoric acid in a weight ratio of 6:3:1, and the amount of curing agent added is 4.0 kg.

[0042] A foaming agent and a curing agent are added to the suspension (suspension 1 or suspension 2), and the mixture is foamed and cured at 70°C to obtain a phenolic foam-nanomontmorillonite composition.

[0043] A foaming agent and a curing agent are added to the suspension 1 and placed in a mold. After foaming and curing at 57.8°C, a phenolic foam-nanomontmorillonite composition T5 is obtained.

[0044] A foaming agent and a curing agent were added to the suspension 2 and placed in a mold. After foaming and curing at 57.8°C, a phenolic foam-nanomontmorillonite composition T6 was obtained.

[0045] The microstructure of the phenolic foam-nanomontmorillonite composition was observed using scanning electron microscopy (SEM), and the results are shown in the figure. Figure 1 The study showed that nano-montmorillonite was uniformly distributed within the foam skeleton. The open-cell ratio of both phenolic foam-nano-montmorillonite compositions was >95%, and statistical analysis of the pore size distribution revealed a pore size range of 150-250 μm.

[0046] C' uses the phenolic foam-nanomontmorillonite composition as the matrix block after cutting, washing and sterilizing.

[0047] The phenolic foam-nanomontmorillonite composition was cut into small pieces of 6 cm × 6 cm × 1.5 cm, repeatedly boiled and washed with deionized water, and then autoclaved at 121℃ to obtain the phenolic foam-nanomontmorillonite matrix. Details are as follows: The phenolic foam-nanomontmorillonite composition T5, after being cut, washed, and sterilized, yields a substrate block T5 for later use.

[0048] The phenolic foam-nanomontmorillonite composition T6, after being cut, washed, and sterilized, yields a substrate block T6 for later use.

[0049] 1.2 Preparation of control matrix block T1 using phenolic foam without the addition of nano-montmorillonite Take 18.95 kg of an aqueous solution of phenolic resin prepolymer, add foaming agent and curing agent, place it in a mold, and foam and cure at 70℃ to produce phenolic foam T1. The types and amounts of foaming agent, foam leveling agent and curing agent are the same as those in the phenolic foam-nanomontmorillonite composition.

[0050] The cured phenolic foam T1 was cut into small pieces of 6 cm × 6 cm × 1.5 cm, repeatedly boiled and washed with deionized water, and then sterilized at 121℃ to obtain the phenolic foam control matrix T1.

[0051] 1.3 at 4 g·L -1 Preparation of control matrix block (CK) using agar solid culture medium At 4 g·L -1 The agar solid culture medium was prepared by weighing 3.2 g of agar, making up to 800 mL of water, autoclaving at 121°C, cooling to 60°C, and pouring into a culture flask to obtain the agar control medium CK.

[0052] 2. Rooting Culture Experimental group T5: The substrate block T5, prepared by the phenolic foam-nanomontmorillonite composition, was saturated with 1 / 2 LM of liquid rooting medium, the specific formula of which is as follows: 825 mg·L⁻¹ -1 NH4NO3, 950 mg·L -1 KNO3, 451.7 mg·L -1 MgSO4, 15.5 mg·L -1 H2BO3, 0.25 mg·L -1 CuSO4·5H2O, 10.5 mg·L -1 MnSO4·H2O, 2.08 mg·L -1 KI, 11 mg·L -1 CaCl2·2H2O, 170 mg·L -1 KH2PO4, 1.13 mg·L -1 Na2SO4·10H2O, 0.085 mg·L -1 CoCl2·8H2O, 18 mg·L -1 EDTANa2, 15 mg·L -1 FeSO4, 21.5 mg·L -1 ZnSO4·7H2O, 100 mg·L -1 Inositol, 0.5 mg·L -1 Niacin, 10.1 mg·L -1 Vitamin B1, 0.1 mg / L -1 Vitamin B6, 8 g·L -1 Sucrose, 4g·L -1 Plant gel and 5 mg·L -1 Glutamine.

[0053] Experimental group T6: The substrate block T6, prepared by the phenolic foam-nanomontmorillonite composition, was saturated with 1 / 2 LM of liquid rooting medium.

[0054] Control group T1: The control substrate block T1, prepared using phenolic foam without added montmorillonite, was saturated with 1 / 2 LM of liquid rooting medium.

[0055] Control group (CK): 4 g·L -1 The control substrate block CK was prepared using agar solid culture medium.

[0056] The four types of substrate blocks were placed in planting containers, and European spruce seedlings with uniform growth were inoculated onto the four types of substrate blocks respectively, with 30 seedlings per treatment and repeated 3 times.

[0057] Under the same conditions (25℃, low light intensity, 15 μmol·m), -2 ·s -1 Cultured for 8 weeks.

[0058] The culture is a solid-liquid composite culture performed under conditions where a shallow liquid layer is maintained at the bottom of the planting container. The bottom of the substrate block is immersed in a shallow liquid layer formed by the liquid rooting medium, but the shallow liquid layer does not submerge the substrate block.

[0059] 3. Results See results Figure 2 .

[0060] The proportion of lateral roots is shown in Figure 2 The lateral root occurrence rate of C:T5 reached 62%, and that of T6 reached 43.75%, both of which were significantly higher than those of the control group CK (0%) and control group T1 (40%).

[0061] See lateral root count Figure 2 The number of lateral roots per seedling in T5 and T6 were 2.4 and 1.5, respectively. Among them, T5 was significantly higher than the control group CK (0 roots) and control group T1 (0.9 roots).

[0062] This invention creatively introduces a low concentration of nano-montmorillonite into a phenolic foam matrix, successfully preparing a novel composite rooting matrix. This matrix not only provides physical support but also actively regulates and significantly promotes the formation of lateral roots in spruce seedlings, effectively solving the industry problem of poor root quality in regenerated seedlings. It has significant value for promoting the development of commercial micropropagation technology for forest trees.

[0063] The present invention has been described in detail above. For those skilled in the art, the invention can be practiced in a wide range of ways with equivalent parameters, concentrations, and conditions without departing from its spirit and scope, and without requiring unnecessary experiments. Although specific embodiments have been given, it should be understood that further modifications can be made to the invention. In summary, according to the principles of the invention, this application is intended to include any changes, uses, or improvements to the invention, including changes made using conventional techniques known in the art that depart from the scope disclosed herein. Some of the essential features can be applied within the scope of the following appended claims.

Claims

1. The application of the composition, characterized in that: The application is any one of Y1-Y3: Application of Y1 in the preparation of porous substrate blocks for rooting of somatic embryos; Application of Y2 in promoting lateral root formation in plants; Application of Y3 in plant cultivation; The composition is made from raw materials including phenolic resin prepolymer and nano-montmorillonite, wherein the mass ratio of the phenolic resin prepolymer to nano-montmorillonite is 18950:360-900, and the phenolic resin prepolymer is a mixture obtained by mixing phenol:formaldehyde:paraformaldehyde:urea:polydicocarbamate:water in a molar ratio of 20:6:12:1:3:

1.

2. The application according to claim 1, characterized in that: The composition is a composition obtained by adding the nano-montmorillonite to the phenolic resin prepolymer, followed by foaming and curing.

3. The application according to claim 2, characterized in that: The composition has an open porosity > 95% and a pore size range of 150 - 250 μm.

4. The application according to claim 2 or 3, characterized in that: The foaming and curing process uses a foaming agent, a foam leveling agent, and a curing agent as additives; the foaming agent is obtained by mixing n-pentane and n-hexane in a weight ratio of 2:1; the foam leveling agent is silicone oil; and the curing agent is obtained by mixing p-toluenesulfonic acid, phenolsulfonic acid, and phosphoric acid in a weight ratio of 6:3:

1.

5. The composition according to any one of claims 1-4.

6. A method for preparing the composition, characterized in that: The composition is the composition of claim 4, and the method includes the following steps: A. Premixing: The nano-montmorillonite is dispersed in the phenolic resin prepolymer to form a suspension; B. Foaming and curing: The foaming agent, the foam leveling agent and the curing agent are added to the suspension, and then foaming and curing are performed to obtain the composition.

7. The method according to claim 6, characterized in that: The foaming and curing were carried out at 57.8°C.

8. A method for preparing cultivation substrate blocks, characterized in that: The matrix block is composed of the composition of claim 5, and the method includes the following steps: The composition is prepared according to the method of claim 6 or 7, and the composition is cut, washed and sterilized to obtain the matrix block.

9. A method for promoting rooting of spruce seedlings with basal embryos, characterized in that: The method includes the step of inoculating spruce seedlings onto a substrate block containing an adsorbed liquid rooting medium for cultivation, wherein the substrate block is the substrate block described in claim 8.

10. The method according to claim 9, characterized in that: During the cultivation process, the bottom of the substrate block is immersed in the liquid rooting medium, but the liquid rooting medium does not submerge the substrate block.

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