Method for inducing rubber tree anther to form fragile embryonic callus by using hydroxylated multi-walled carbon nanotubes

Abstract

The invention provides a method for inducing a rubber tree anther to form a fragile embryonic callus by using a hydroxylated multi-walled carbon nanotube. The method specifically comprises the following steps: 1) adding a multi-walled carbon nanotube raw material into a concentrated nitric acid-concentrated sulfuric acid mixed system, and carrying out constant-temperature ultrasonic treatment for 3-5 hours at the temperature of 55-65 DEG C, the power of 200-400 W and the frequency of 30-50 kHz to prepare MWCNTs-OH with the hydroxylation degree of 8-12%; 2) HCK-12 is taken as a basic culture medium, 20-160mg / LMWCNTs-OH is added, the pretreated mononuclear late-stage anther is subjected to dark culture at 24-26 DEG C for 42 days, the callus induction rate can reach 100%, and the proportion of fragile embryonic callus can reach 82.8%; and (3) by taking HE-7 as a basic culture medium, adding 5-40mg / L LMWCNTs-OH, and culturing the callus for 42 days under the same condition, so that the embryo yield can reach 88%. And the normal embryo rate is as high as 70%. Through cooperation of the hydroxylated multi-walled carbon nanotubes and the specific culture medium, the problems that in the prior art, the embryogenic callus proportion is low, and hormone and an anti-browning agent interfere with each other are effectively solved, and an efficient and stable method is provided for rubber tree anther culture.

Description

Technical Field

[0001] This invention belongs to the field of plant tissue culture technology, specifically relating to a method for inducing the formation of fragile embryogenic callus from rubber tree anthers using hydroxylated multi-walled carbon nanotubes. Background Technology

[0002] As an important industrial raw material crop, the cultivation and propagation of superior rubber varieties are crucial to the development of the rubber industry. Anther tissue culture is one of the core methods for improving rubber tree varieties and rapid propagation. However, in practical applications, this technology generally faces bottlenecks such as poor callus quality and insufficient supply of materials for subsequent research. Specifically, the callus formed by the dedifferentiation of rubber tree anthers is mostly a dense structure, and embryogenic and non-embryonic callus tissues are mixed, making effective separation and purification difficult. In addition, this type of dense callus tissue usually reaches its maximum growth after 50 days of culture, after which it gradually enters the senescence stage and eventually dies. This characteristic makes related research progress slowly due to a lack of materials.

[0003] In stark contrast, fragile embryogenic callus, due to its ability to undergo long-term subculture, has gradually become an ideal material for promoting rubber tree tissue culture research and related biotechnological applications. Therefore, developing a method to rapidly induce fragile embryogenic callus has become a key problem urgently needing to be solved in this field. Research findings from France, India, and other countries indicate that adding silver nitrate to the rubber tree callus culture medium, or using high-calcium, high-sucrose culture conditions, followed by 2-3 subcultures, can improve the structure of the callus to some extent, making it more loose and fragile.

[0004] However, there is still considerable room for improvement in these existing technologies in terms of induction efficiency and enhancement of embryogenic expression: on the one hand, the induction efficiency of existing methods is low, and structural improvement can only be achieved through multiple subcultures, which is time-consuming; on the other hand, conventional treatments have limited effect on improving the embryogenicity of callus and cannot meet the demand for high-quality embryogenic callus for subsequent embryogenesis and plant regeneration.

[0005] In view of this, the present invention is hereby proposed. Summary of the Invention

[0006] The purpose of this invention is to provide a method for inducing the formation of fragile embryogenic callus from rubber tree anthers using hydroxylated multi-walled carbon nanotubes (MWCNTs). Through extensive experiments, the research team discovered that designing the culture medium components and adding specific amounts of hydroxylated MWCNTs significantly improves callus structure during rubber tree anther tissue culture. Specifically, the synergistic effect of hydroxylated MWCNTs and culture medium components effectively promotes the transformation of callus induced from rubber tree anthers into fragile embryogenic callus. Simultaneously, it significantly improves the induction efficiency of rubber tree anther callus and enhances its embryogenicity. During the embryogenesis stage, it effectively increases the embryogenesis rate of rubber tree anther callus.

[0007] To achieve the above objectives, the present invention provides a method for inducing the formation of fragile embryogenic callus from rubber tree anthers using hydroxylated multi-walled carbon nanotubes, comprising the following steps:

[0008] (1) Preparation of hydroxylated multi-walled carbon nanotubes: Add multi-walled carbon nanotube raw material to a mixture of concentrated nitric acid and concentrated sulfuric acid, and react with ultrasonication at a constant temperature of 55-65℃ for 3-5 hours. After the reaction is completed, wash with deionized water until the washing solution is neutral, and dry to obtain multi-walled carbon nanotubes with a degree of hydroxylation of 8-12%.

[0009] (2) Induction of fragile embryogenic callus: HCK-12 was used as the basal medium with the following components and concentrations: 50 ml / L MS1, 10 ml / L MS2, 10 ml / L MS3, 10 ml / L MS4, 15 ml / L KT, 15 ml / L NAA, 15 ml / L 2,4-D, 0.1 g / L inositol, 0.3 g / L aspartic acid, 70 g / L sucrose, 50 ml / L coconut water, 2.2 g / L plant gel, and 20-160 mg / L hydroxylated multi-walled carbon nanotubes prepared in step (1). The pH was adjusted to 5.7-5.9, and after high-temperature sterilization, the rubber tree anthers were inoculated at room temperature for culture.

[0010] (3) Induction of callus embryogenesis: HE-7 as the basal culture medium, with the following components and concentrations: 40 ml / L MS1, 10 ml / L MS2, 10 ml / L MS3, 10 ml / L MS4, 10 ml / L 6-benzylaminopurine, 30 ml / L KT, 0.6 ml / L 2,4-D, 5 ml / L gibberellin, 50 ml / L coconut water, 0.1 g / L inositol, 1 g / L activated carbon, 70 g / L sucrose, 2.2 g / L plant gel, and 5-40 mg / L hydroxylated multi-walled carbon nanotubes prepared in step (1). After adjusting the pH to 5.7-5.9 and sterilizing at high temperature, the callus obtained in step (2) was inoculated at room temperature for culture.

[0011] In a preferred embodiment, in step (1), the volume ratio of concentrated nitric acid to concentrated sulfuric acid is 1:3.

[0012] In a preferred embodiment, in step (1), the concentrated nitric acid has a mass fraction of 65-68% and the concentrated sulfuric acid has a mass fraction of 95-98%.

[0013] In a preferred embodiment, in step (1), the ratio of the multi-walled carbon nanotube raw material to the mixed acid (concentrated nitric acid and concentrated sulfuric acid) is 1g:40ml.

[0014] In a preferred embodiment, in step (1), the multi-walled carbon nanotube raw material has a diameter of 10-20 nm, a length of 1-5 μm, and a purity of ≥98%.

[0015] In a preferred embodiment, in step (1), the ultrasonic conditions include: power of 200-400W and frequency of 30-50kHz.

[0016] In a preferred embodiment, in step (1), the ultrasonic reaction conditions are: ultrasonic power of 300W, frequency of 40kHz, and constant temperature ultrasonication at 60℃ for 4 hours. To maintain the constant temperature, an ice bath can be used for temperature control.

[0017] Through extensive experiments, the inventors discovered that low hydroxylation (<8%) leads to poor dispersibility and insufficient cell affinity, while high hydroxylation (>12%) increases the risk of carbon nanotube structure breakage. Therefore, in this invention, by controlling the processing temperature of around 60°C, ultrasonic conditions, the ratio of multi-walled carbon nanotubes to mixed acid, and the volume ratio of concentrated nitric acid to concentrated sulfuric acid in the mixed acid, multi-walled carbon nanotubes with a hydroxylation degree of 8-12% were obtained, effectively shortening the reaction time and reducing reaction energy consumption.

[0018] In a preferred embodiment, in step (1), the washing solution is neutral with a pH of 6.8-7.2.

[0019] In a preferred embodiment, in step (1), the drying reaction conditions are: drying at 40-60°C for 6-8 hours.

[0020] In a preferred embodiment, in step (2), the rubber tree anthers are pretreated uninucleate marginal stage anthers, and the specific sampling method includes:

[0021] S1 uses microscopic examination to select pollen grains at the single cell stage where the cell nucleus is squeezed to the edge and the development is uniform, and removes deformed, browned or diseased anthers.

[0022] S2 Place the screened anthers in a sterile Erlenmeyer flask and rinse with sterile water 1-3 times, 0.5-2 minutes each time, to remove surface dust;

[0023] S3. Place the treated anthers in 75% medical alcohol (by volume) and soak for 20-30 seconds.

[0024] S4. Remove the anthers and place them in a 0.1% sodium hypochlorite solution for 10-12 minutes, gently shaking once every 2 minutes during this time.

[0025] S5 removes the flower extract and rinses it repeatedly with sterile water 5-6 times, 1-2 minutes each time, to remove residual disinfectant.

[0026] In a preferred embodiment, in step (2), the concentration of the hydroxylated multi-walled carbon nanotubes is 20 mg / L, 40 mg / L, 80 mg / L, or 160 mg / L.

[0027] In a preferred embodiment, in step (2), the high-temperature sterilization conditions include: sterilization at 121°C for 20 minutes.

[0028] In a preferred embodiment, in step (2), the culture conditions include: culture in complete darkness at 24-26°C for 35-42 days.

[0029] In a preferred embodiment, in step (2), the induction rate of the fragile embryogenic callus can reach 100%.

[0030] In a preferred embodiment, in step (3), the concentration of the hydroxylated multi-walled carbon nanotubes is 5 mg / L, 10 mg / L, 20 mg / L, or 40 mg / L.

[0031] In a preferred embodiment, in step (3), the high-temperature sterilization conditions include: sterilization at 121°C for 20 minutes.

[0032] In a preferred embodiment, in step (3), the culture conditions include: culture in complete darkness at 24-26°C for 35-42 days.

[0033] In a preferred embodiment, in step (3), the embryogenesis rate of the fragile embryogenic callus can reach 88%.

[0034] Compared with the prior art, the technical solution of the present invention has the following advantages:

[0035] (I) In the field of rubber tree anther callus induction, most existing technologies rely on the natural effects of culture medium components on cells, which is a passive induction mode. This mode makes it difficult to precisely control the direction of embryogenic differentiation, often resulting in a high proportion of dense non-embryonic callus and a tendency for embryogenic cells to age.

[0036] In this invention, a synergistic system of HCK-12-specific culture medium and hydroxylated multi-walled carbon nanotubes was designed to achieve a shift from passive induction to active regulation. At the molecular and cellular level, the hydroxyl groups on the surface of the hydroxylated multi-walled carbon nanotubes can target and adsorb hormones such as KT, NAA, and 2,4-D in the culture medium via hydrogen bonds, preventing excessive cell division (a major cause of non-embryonic differentiation) caused by excessively high hormone concentrations in the early stages of culture. Simultaneously, in the later stages of culture, these hormones are slowly released, maintaining a stable environment of low hormone concentration in the anther cells. This stable environment activates intracellular signaling pathways related to embryogenic differentiation, such as promoting calcium ion entry into the cell through cell membrane channels, increasing intracellular calcium ion concentration, and subsequently upregulating the expression of embryogenic genes such as LEC1 and ABI3, thus driving cell differentiation towards embryogenicity. Furthermore, the hydroxylated multi-walled carbon nanotubes have better dispersibility, allowing them to be evenly distributed in the intercellular spaces. Through physical separation, they prevent cell clusters from tightly agglomerating to form dense callus, while also scavenging reactive oxygen species produced by cell metabolism, reducing apoptosis, and prolonging the survival period of embryogenic cells. The above-mentioned mechanisms synergistically solve the problems of low embryogenic induction efficiency and dense callus structure in existing technologies, providing high-quality embryogenic materials for subsequent embryo culture.

[0037] (ii) During the embryogenesis stage of rubber tree callus tissue, existing technologies generally suffer from interference between hormones and anti-browning agents. For example, although activated carbon can adsorb browning substances, it can also easily adsorb hormones in the culture medium, leading to fluctuations in hormone concentration during the embryogenesis stage, which in turn causes asynchronous embryoid differentiation and a high rate of deformities.

[0038] To address this, this invention achieves a synergistic effect of ordered maturation and anti-browning by optimizing the composition of HE-7 culture medium and regulating the concentration of hydroxylated multi-walled carbon nanotubes. From a metabolic and developmental perspective, firstly, the hormone combination of low concentrations of 2,4-D (0.6 ml / L) and GA3 (5 ml / L) in HE-7 culture medium, combined with the down-regulation of hydroxylated multi-walled carbon nanotube concentrations (5-40 mg / L) during embryogenesis, can promote the transition of embryoids from a dedifferentiated state to a differentiated and mature state: low concentrations of 2,4-D can prevent callus tissue from re-entering the dedifferentiated state, while GA3 can promote organ development of embryoids, and hydroxylated multi-walled carbon nanotubes can bind to GA3 through their surface hydroxyl groups, increasing the enrichment of GA3 around cells and accelerating the maturation process of embryoids. Secondly, hydroxylated multi-walled carbon nanotubes preferentially occupy adsorption sites on the surface of activated carbon through competitive adsorption, reducing the adsorption of key hormones such as 6BA and KT by activated carbon. Simultaneously, the carbon nanotubes themselves can directly remove substances related to browning, such as H2O2 produced by cell metabolism, forming a dual anti-browning mechanism with activated carbon and reducing the browning rate of callus tissue. Furthermore, hydroxylated multi-walled carbon nanotubes can synergistically interact with sucrose and aspartic acid, providing a stable supply of nutrients during embryoid development and preventing developmental arrest due to nutrient imbalance. Through these regulatory mechanisms, the embryo emergence rate ultimately reaches 88%, with highly uniform embryoid maturity, providing high-quality materials for subsequent plant regeneration. Detailed Implementation

[0039] To enable those skilled in the art to better understand the present invention, the present invention will be further described in detail below with reference to specific embodiments. However, it should be understood that the scope of protection of the present invention is not limited to the specific embodiments. Based on the embodiments in this application, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this application.

[0040] Unless otherwise specified, the technical means used in this invention are conventional means well known to those skilled in the art. All raw materials, reagents, instruments, and equipment used in this invention can be purchased commercially or prepared using existing methods. Unless otherwise specified, all reagents used in this invention are of analytical grade. In this invention, the room temperature is 25±2℃.

[0041] In this embodiment of the invention, the proportion of fragile embryonic callus is calculated by the following method:

[0042] Sample preparation: 30 independent callus tissues were randomly selected from each group as test samples and placed in sterile culture dishes in a laminar flow hood for later use.

[0043] Morphological screening: Embryogenic callus was selected by observation with a stereomicroscope with magnification of 10-40x: embryogenic callus is pale yellow or milky white, loose in texture, with a 0.2-0.5 mm granular structure on the surface, and has low adhesion to the culture dish wall; non-embryogenic callus that is dark brown / green, dense in texture, smooth or blocky on the surface was excluded.

[0044] Fragility verification: Use sterile forceps to gently pick up a single embryogenic callus and transfer it to sterile filter paper. Gently shake the filter paper and observe whether it naturally disperses into more than 3 pale yellow / milky white particles of 0.1-0.3 mm. Those that can disperse naturally are judged as fragile embryogenic callus. Count the number of fragile embryogenic callus in each group (denoted as A).

[0045] Calculation formula: The number of successful inductions in each group in Table 1 (denoted as B) is calculated according to the formula "Percentage of fragile embryo callus (%) = (A / B) × 100", and the average value is taken as the final result.

[0046] In this embodiment of the invention, the browning rate is calculated by the following method:

[0047] Sample preparation: 30 independent callus tissues were randomly selected from each group as test samples and placed in sterile culture dishes in a laminar flow hood for later use.

[0048] Judgment criteria: Dark brown or brownish-brown areas appear on the surface or cross-section of the callus, and the area of ​​the brown area is ≥10% (e.g., if the diameter of the callus is 1cm, the diameter of the brown area is ≥0.32cm, which is considered browning); if the entire callus is dark brown or black necrotic spots appear, it is directly judged as browning.

[0049] Calculation formula: Browning rate (%) = (Number of browned calluses / Total number of calluses to be tested) × 100, take the average value.

[0050] Example 1

[0051] The preparation of hydroxylated multi-walled carbon nanotubes includes the following steps:

[0052] A mixed acid was prepared by mixing 65% (w / w) concentrated nitric acid and 98% (w / w) concentrated sulfuric acid at a volume ratio of 1:3. Carbon nanotubes were added to the mixed acid at a mass-to-volume ratio of 1g of ordinary multi-walled carbon nanotubes to 40ml of the mixed acid. The mixture was placed in a 60℃ constant-temperature water bath and ultrasonically reacted for 4 hours at a power of 300W and a frequency of 40kHz. During the reaction, the system temperature rose, requiring the addition of ice to control the temperature. After the reaction, the product was repeatedly washed with deionized water until the pH of the washing solution reached 7. The product was then dried in a 50℃ vacuum drying oven for 7 hours to obtain hydroxylated multi-walled carbon nanotubes (MWCNTs-OH) with a hydroxylation degree of 10%.

[0053] Example 2

[0054] Induction of fragile embryogenic callus includes the following steps:

[0055] Selection and pretreatment of anthers from S1 rubber trees: Inflorescences of the Brazilian rubber tree variety Reyan 73397 during its full bloom period were selected. Anthers in the uninucleate marginal stage (cell nuclei pushed towards the edge and uniformly developed) were screened under a stereomicroscope, and deformed / browned anthers were removed. The anthers were first rinsed three times with sterile water for one minute each time, then soaked in 75% alcohol for 25 seconds, followed by soaking in 0.1% sodium hypochlorite solution for 11 minutes, with gentle shaking every two minutes. Finally, the anthers were rinsed five times with sterile water for two minutes each time.

[0056] S2 fragile embryogenic callus induction: The basal medium was HCK-12, with the following components and concentrations: 50 ml / L MS1, 10 ml / L MS2, 10 ml / L MS3, 10 ml / L MS4, 15 ml / L KT, 15 ml / L NAA, and 15 ml / L... 2,4-D, 0.1 g / L inositol, 0.3 g / L aspartic acid, 70 g / L sucrose, 50 ml / L coconut water, 2.2 g / L plant gel, and 0 mg / L, 20 mg / L, 40 mg / L, 80 mg / L, 160 mg / L, and 200 mg / L of the common multi-walled carbon nanotube raw material (MWCNTs) and the prepared hydroxylated multi-walled carbon nanotubes (MWCNTs-OH) from Example 1, respectively, were added. The pH was adjusted to 5.7-5.9, sterilized at 121℃ for 20 minutes, and then cooled to 25℃ before inoculating the rubber tree anthers from step (1) for culture. The culture conditions were: cultured in a dark environment at 25℃ for 42 days, and photographs were taken on the 42nd day. Each group was repeated 3 times. The specific groups, concentrations, and results are shown in Table 1.

[0057] Table 1

[0058]

[0059] Results and Discussion: As shown in Table 1, after uniformly inoculating 75 anthers, in the MWCNTs group, the effects of each concentration group with added carbon nanotubes were superior to the group with a concentration of 0 mg / L. Within the concentration range of 20-160 mg / L, the callus induction rate was 93.33-96.00%, reaching the highest value (96.00%) at 80 mg / L. The proportion of fragile embryogenic callus was 40.0-48.0%, also peaking at 48.0% at a concentration of 80 mg / L. The browning rate varied between 13.0-30.0%, with the lowest browning rate (13.0%) at 80 mg / L. However, when the concentration increased to 200 mg / L, all indicators decreased, the callus induction rate dropped to 93.33%, the proportion of fragile embryogenic callus dropped to 35.0%, and the browning rate increased to 30.0%. It can be seen that high concentrations of unmodified MWCNTs can have adverse effects on anther cells, such as causing aggregation and interfering with normal cell growth.

[0060] In the MWCNTs-OH group, all concentration groups showed better performance than the 0 mg / L group, and were significantly superior to the MWCNTs group at the same concentration. Within the concentration range of 20-160 mg / L, the callus induction rate was 97.33-99.33%, peaking at 99.33% at 80 mg / L. The proportion of fragile embryogenic callus was 65.0-88.0%, reaching a maximum of 88.0% at 80 mg / L. The browning rate was as low as 5.0-12.0%, only 5.0% at 80 mg / L. Even at a concentration of 200 mg / L, the callus induction rate of the MWCNTs-OH group (96.00%) was still higher than that of the MWCNTs group at the same concentration, further confirming that hydroxylation modification enhances the biocompatibility and induction effect of carbon nanotubes. However, compared to low and medium concentrations, its fragile embryogenic callus proportion and browning rate were generally lower, but still superior to unmodified MWCNTs at the same concentration.

[0061] In summary, the addition of carbon nanotubes (MWCNTs) significantly promotes the induction of callus tissue in rubber tree anthers, with hydroxylated MWCNTs-OH showing a significantly better effect than unmodified MWCNTs. The optimal induction effect is particularly evident at a concentration of 80 mg / L, effectively increasing the callus induction rate and the proportion of fragile embryogenic callus, while reducing the browning rate, thus creating superior conditions for rubber tree anther culture.

[0062] Example 3

[0063] Callus formation includes the following steps:

[0064] S1 uses HE-7 as the basal culture medium, with the following components and concentrations: 40 ml / L MS1, 10 ml / L MS2, 10 ml / L MS3, 10 ml / L MS4, 10 ml / L 6-benzylaminopurine, 30 ml / L KT, 0.6 ml / L 2,4-D, 5 ml / L gibberellin, 50 ml / L coconut water, 0.1 g / L inositol, 1 g / L activated carbon, 70 g / L sucrose, 2.2 g / L plant gel, and 5 mg / L, 10 mg / L, 20 mg / L, and 40 mg / L of the common multi-walled carbon nanotube raw material (MWCNTs) and the prepared hydroxylated multi-walled carbon nanotubes (MWCNTs-OH) from Example 1. The pH was adjusted to 5.7-5.9, sterilized at 121°C for 20 minutes, and then cooled for later use.

[0065] S2 took fragile embryogenic callus tissue cultured for 42 days in Example 2, cut it into 0.5cm × 0.5cm pieces, and inoculated it into the culture medium prepared in S1 at 25℃. It was cultured in the dark at 25℃ for 42 days, and photographs were taken on the 42nd day. Each group was repeated 3 times. The specific groups, concentrations and results are shown in Table 2.

[0066] Table 2

[0067]

[0068] Results and Discussion: The highest embryogenesis rate in the MWCNTs-induced group was only 64.00% (MWCNTs group 5), with a normal embryogenesis rate below 44% and a fresh weight of less than 16 mg per normal embryo. Specifically, the MWCNTs group 6 (induced in Example 2 at 200 mg / L) had an embryogenesis rate of only 45.33%, a normal embryogenesis rate of 26.92%, and a fresh weight of 10.2 mg / embryo. The reason for this may be that the high MWCNT concentration during the induction phase of Example 2 resulted in callus tissue with "low embryogenicity and poor cell viability," making it difficult to achieve efficient embryogenesis even with a reduced concentration during the embryogenesis phase.

[0069] In contrast, the MWCNTs-OH induced groups, at embryogenic concentrations of 5-40 mg / L, showed embryogenic rates of 81.33-88.00%, normal embryo rates of 60.00-70.00%, and fresh weights of individual normal embryos of 18.6-22.2 mg. Compared to the MWCNTs groups at the same embryogenic concentration, the embryogenic rate increased by 29.9-60.0%, and the normal embryo rate increased by 48.5-85.2%. This demonstrates that the present invention produces a synergistic effect in both callus induction and embryogenic stages: during the induction stage, the fragile embryogenic callus cells formed by MWCNTs-OH exhibit strong differentiation potential; during the embryogenic stage, MWCNTs-OH further promotes orderly development of the radicle and cotyledons and reduces malformations by adsorbing gibberellins and 6-benzylaminopurine. Even in the MWCNTs-OH group induced at 200 mg / L, the embryogenic rate (56.00%) and normal embryo rate (47.95%) were still higher than those in the MWCNTs groups at the same concentration, further validating the important role of hydroxylation modification.

[0070] The foregoing description of specific exemplary embodiments of the invention is for illustrative and explanatory purposes. These descriptions are not intended to limit the invention to the precise forms disclosed, and it will be apparent that many changes and variations can be made in accordance with the foregoing teachings. The exemplary embodiments were chosen and described in order to explain the specific principles of the invention and its practical application, thereby enabling those skilled in the art to implement and utilize various different exemplary embodiments of the invention, as well as various different choices and variations. The scope of the invention is intended to be defined by the claims and their equivalents.

Claims

1. A method for inducing the formation of fragile embryogenic callus from rubber tree anthers using hydroxylated multi-walled carbon nanotubes, characterized in that, Includes the following steps: (1) Preparation of hydroxylated multi-walled carbon nanotubes: Add multi-walled carbon nanotube raw material to a mixture of concentrated nitric acid and concentrated sulfuric acid, and react with ultrasonication at a constant temperature of 55-65℃ for 3-5 hours. After the reaction is completed, wash with deionized water until the washing solution is neutral, and dry to obtain multi-walled carbon nanotubes with a degree of hydroxylation of 8-12%. (2) Induction of fragile embryogenic callus: HCK-12 was used as the basal medium with the following components and concentrations: 50 ml / L MS1, 10 ml / L MS2, 10 ml / L MS3, 10 ml / L MS4, 15 ml / L KT, 15 ml / L NAA, 15 ml / L 2,4-D, 0.1 g / L inositol, 0.3 g / L aspartic acid, 70 g / L sucrose, 50 ml / L coconut water, 2.2 g / L plant gel, and 20-160 mg / L hydroxylated multi-walled carbon nanotubes prepared in step (1). The pH was adjusted to 5.7-5.9, and after high-temperature sterilization, the rubber tree anthers were inoculated at room temperature for culture. (3) Induction of callus embryogenesis: HE-7 as the basal culture medium, with the following components and concentrations: 40 ml / L MS1, 10 ml / L MS2, 10 ml / L MS3, 10 ml / L MS4, 10 ml / L 6-benzylaminopurine, 30 ml / L KT, 0.6 ml / L 2,4-D, 5 ml / L gibberellin, 50 ml / L coconut water, 0.1 g / L inositol, 1 g / L activated carbon, 70 g / L sucrose, 2.2 g / L plant gel, and 5-40 mg / L hydroxylated multi-walled carbon nanotubes prepared in step (1). After adjusting the pH to 5.7-5.9 and sterilizing at high temperature, the callus obtained in step (2) was inoculated at room temperature for culture.

2. The method for inducing the formation of fragile embryogenic callus from rubber tree anthers using hydroxylated multi-walled carbon nanotubes as described in claim 1, characterized in that, In step (1), the volume ratio of concentrated nitric acid to concentrated sulfuric acid is 1:3; The ratio of the multi-walled carbon nanotube raw material to the mixed acid is 1g:40ml; The multi-walled carbon nanotube raw material has a diameter of 10-20 nm, a length of 1-5 μm, and a purity of ≥98%.

3. The method for inducing the formation of fragile embryogenic callus from rubber tree anthers using hydroxylated multi-walled carbon nanotubes as described in claim 1, characterized in that... In step (1), the ultrasonic conditions include: power of 200-400W and frequency of 30-50kHz.

4. The method for inducing the formation of fragile embryogenic callus from rubber tree anthers using hydroxylated multi-walled carbon nanotubes as described in claim 1, characterized in that... In step (2), the rubber tree anthers are pretreated uninucleate marginal stage anthers, and the specific sampling method includes: S1 uses microscopic examination to select pollen grains at the single cell stage where the cell nucleus is squeezed to the edge and the development is uniform, and removes deformed, browned or diseased anthers. S2. Place the screened anthers in a sterile Erlenmeyer flask and rinse with sterile water 1-3 times, 0.5-2 minutes each time, to remove surface dust. S3. Place the treated anthers in 75% medical alcohol (by volume) and soak for 20-30 seconds. S4. Remove the anthers and place them in a 0.1% sodium hypochlorite solution for 10-12 minutes, gently shaking once every 2 minutes during this time. S5 removes the flower extract and rinses it repeatedly with sterile water 5-6 times, 1-2 minutes each time, to remove residual disinfectant.

5. The method for inducing the formation of fragile embryogenic callus from rubber tree anthers using hydroxylated multi-walled carbon nanotubes as described in claim 1, characterized in that... In step (2), the concentration of the hydroxylated multi-walled carbon nanotubes is 20 mg / L, 40 mg / L, 80 mg / L, and 160 mg / L.

6. The method for inducing the formation of fragile embryogenic callus from rubber tree anthers using hydroxylated multi-walled carbon nanotubes as described in claim 1, characterized in that, In step (2), the culture conditions include: culture in complete darkness at 24-26℃ for 35-42 days.

7. The method for inducing the formation of fragile embryogenic callus from rubber tree anthers using hydroxylated multi-walled carbon nanotubes as described in claim 1, characterized in that, In step (2), the induction rate of the fragile embryogenic callus can reach 100%.

8. The method for inducing the formation of fragile embryogenic callus from rubber tree anthers using hydroxylated multi-walled carbon nanotubes as described in claim 1, characterized in that... In step (3), the concentration of the hydroxylated multi-walled carbon nanotubes is 5 mg / L, 10 mg / L, 20 mg / L, or 40 mg / L.

9. The method for inducing the formation of fragile embryogenic callus from rubber tree anthers using hydroxylated multi-walled carbon nanotubes as described in claim 1, characterized in that, In step (3), the culture conditions include: culture in complete darkness at 24-26℃ for 35-42 days.

10. The method for inducing the formation of fragile embryogenic callus from rubber tree anthers using hydroxylated multi-walled carbon nanotubes as described in claim 1, characterized in that, In step (3), the embryogenesis rate of the fragile embryogenic callus can reach 88%.

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