A method for rapid propagation of monocotyledonous plant monstera deliciosa

Abstract

The present application relates to a kind of monocotyledon green wisteria rapid propagation method, using the characteristics of green wisteria specific tissue cell totipotency, under open condition, using liquid medium to induce green wisteria specific site of in vitro leaf split differentiation, produce regenerative root and regenerative bud, finally form plant.The present application aims at the rapid propagation method of green wisteria leaf, without sterile environment, convenient to take material, easy to operate, low in cost, short in period, high in survival rate, with the advantages of high efficiency, economy, easy to scale, factory, etc., in green wisteria rapid seedling, specific material preservation and propagation has greater application value.

Description

Technical Field

[0001] This invention relates to a method for rapidly propagating the monocotyledonous plant *Epipremnum aureum*, belonging to the field of rapid plant propagation technology. Background Technology

[0002] Pothos (Scindapsus aureus) is an evergreen vine belonging to the genus Scindapsus in the family Araceae. It is a monocotyledonous plant. It has high ornamental and ecological value in terms of indoor and outdoor viewing, landscaping, air purification, and water purification.

[0003] Currently, the main propagation method for pothos is cuttings, which has drawbacks such as low propagation coefficient, slow growth rate, and poor genetic stability. Tissue culture is an effective way to achieve rapid propagation of pothos. Recent research has mainly used pothos stem segments as explants to obtain regenerated pothos plants through callus differentiation or direct induction of shoots. However, due to the limitations of explant selection and the complexity of the effects of plant growth regulators on regeneration, current pothos tissue culture still suffers from low regeneration frequency, high cost, and relatively high technical requirements for operators. To meet the large demand for pothos from socio-economic development and overcome the shortcomings of existing propagation methods, it is necessary to develop a rapid, effective, and convenient method for rapid propagation of pothos.

[0004] Studies have shown that some plants can achieve non-tissue culture regeneration under open conditions, such as leaf cuttings. Leaf cuttings utilize the regenerative capacity of a plant's leaves or parts thereof for propagation. Adventitious buds and roots develop at the base, edge, or center of the cutting, forming new individual plants. Leaf cuttings are easy to perform, have a high survival rate, are low in cost, and produce seedlings with intact root systems and uniform emergence. Currently, only a few plants, such as those in the genera *Begonia*, *Peperomia*, *Gesneriaceae*, and monocotyledonous plants like *Sansevieria trifasciata*, can be propagated by leaf cuttings. Non-tissue culture regeneration of *Epipremnum aureum* under open conditions has not yet been reported. Summary of the Invention

[0005] To address the shortcomings of existing technologies, the purpose of this invention is to provide a method for rapidly propagating the monocotyledonous plant *Epipremnum aureum*. Utilizing the totipotency of specific cell tissues in *Epipremnum aureum*, a liquid culture medium is used under open conditions to induce the division and differentiation of specific parts of detached leaves, producing regenerated roots and shoots, ultimately forming a plant.

[0006] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0007] A method for rapidly propagating the monocotyledonous plant *Epipremnum aureum* includes the following steps:

[0008] (1) Cut healthy, disease-free leaves from the main stem or side branches of the mother plant, which are 10-40 days old, and are free from obvious pests and diseases.

[0009] (2) Processing detached pothos leaves: After washing, trim the petioles and cut them into wedge shapes;

[0010] (3) Cultivate the treated detached pothos leaves: Place the leaf surface on a foam board and immerse the petiole in a liquid culture medium for cultivation;

[0011] (4) After the detached leaves have taken root and sprouted, remove the detached leaves and cultivate them in soil, and manage them in a regular manner.

[0012] The method for cutting pothos leaves is as follows: use a sharp double-edged blade to quickly cut the leaf from bottom to top, with the cutting point located at the lower part of the petiole, 2-3 mm away from the stem; the detached pothos leaf does not contain axillary buds or adjacent tissues.

[0013] Specific steps for processing detached pothos leaves:

[0014] 1) Cleaning: First, carefully clean the entire leaf with clean tap water 2-3 times, then wipe the entire leaf with medical absorbent cotton moistened with distilled water 2-3 times, and finally clean the entire leaf with distilled water 2-3 times.

[0015] 2) Pruning: Use a sharp double-edged blade to quickly cut the petiole into a wedge shape. Expose the pruned petiole to the air in a shady place for 3-4 minutes. Avoid damaging the leaves throughout the process.

[0016] If the prepared leaves cannot be hydroponically grown immediately, wrap the leaves with clean, moist filter paper, wrap the petiole with moist medical absorbent cotton, place them in a seed germination box, and store them in a dark place for later use.

[0017] Steps for culturing detached pothos leaves after treatment:

[0018] 1) Pour 1 / 4 volume of liquid culture medium into the seed germination box and place a foam board on the surface of the liquid culture medium;

[0019] 2) Place the treated detached pothos leaves on a foam board and immerse the petioles in liquid culture medium; place the seed germination box in a clean self-sealing bag, leaving the bag open; the culture temperature is 24±2℃, the light intensity is 800lx, and the light duration is 8h / d.

[0020] The liquid culture medium was prepared by adding PVP to 1 / 8 MS basal liquid culture medium at a concentration of 500 mg / L and a pH of 6.0. The specific composition was: 237.5 mg / L KNO3, 206.25 mg / L NH4NO3, 21.25 mg / L KH2PO4, 46.25 mg / L MgSO4·7H2O, 55 mg / L CaCl2·2H2O, 0.104 mg / L KI, 0.775 mg / L H3BO3, 2.79 mg / L MnSO4·4H2O, 1.075 mg / L ZnSO4·7H2O, 0.03125 mg / L Na2MoO4·2H2O, 0.003125 mg / L CuSO4·5H2O, 0.003125 mg / L CoCl2, and 4.6625 mg / L MgSO4·7H2O. Na2·EDTA·2H2O, 3.475 mg / L FeSO4·7H2O, 12.5 mg / L inositol, 0.25 mg / L glycine, 0.0125 mg / L VB1, 0.0625 mg / L VB6, 0.0625 mg / L VB5.

[0021] During the cultivation of detached pothos leaves, the liquid culture medium was changed twice, and then once every 20 days; when cultivating detached leaves in soil, garden soil was used as the substrate.

[0022] Beneficial effects of this invention:

[0023] 1. The method for regenerating pothos leaves of this invention utilizes the totipotency of specific tissue cells in pothos. Under open conditions, a special hydroponic solution is used to induce the division and differentiation of specific parts of detached pothos leaves, producing regenerated roots and shoots, ultimately forming a plant. This method does not require a sterile environment, is easy to obtain materials, simple to operate, low in cost, short in cycle, and has a high survival rate. It has the advantages of high efficiency, economy, and ease of large-scale and industrial production; the success rate of regenerated seedlings reaches over 88%, and the survival rate of regenerated plants after transplanting reaches over 95%. This method has significant application value in the fields of rapid pothos seedling cultivation, preservation and propagation of specific materials.

[0024] 2. The in vitro leaf regeneration system for *Epipremnum aureum* established in this invention differs significantly from previous traditional tissue culture regeneration systems. This regeneration pathway involves direct induction of adventitious buds from explants, resulting in better genetic stability. Plant tissue culture typically involves explant dedifferentiation to produce callus, which then differentiates into buds, eventually leading to root formation. However, root formation is often more difficult than bud formation. During callus culture, there is a certain probability of mutations occurring, leading to inconsistent genetic characteristics, and not all callus tissues possess the regenerative capacity to produce new seedlings.

[0025] In this invention, under open conditions, the general process of regeneration of detached leaves of pothos is as follows: first, adventitious roots are formed, followed by adventitious buds. This regeneration system has distinct characteristics: the adventitious roots absorb nutrients and water, while the detached leaves perform photosynthesis, forming a relatively independent and complete regeneration system, ensuring high efficiency in regeneration.

[0026] 3. This invention utilizes a liquid culture medium to cultivate pothos, thus improving the regeneration rate. Plant culture media are generally divided into solid and liquid media. Solid media are the most commonly used method for cultivating plant materials. Although this method is simple and easy to implement, nutrient distribution is uneven, growth rates are inconsistent, and browning and poisoning often occur. Liquid media have better fluidity, allowing for more precise control of solution temperature, nutrient concentration, and gas content. Furthermore, liquid media can more quickly dilute and disperse browning products produced by explants, reducing the content and concentration of toxic substances around the explants and effectively mitigating their toxic effects. Another advantage of liquid media is its cost-effectiveness and efficiency. Compared to solid media, liquid media requires less adhesion from plant tissues, allowing them to suspend and maintain close contact. This stimulates and promotes the absorption of nutrients and hormones, resulting in a better nutritional and growth environment, thereby promoting rapid growth and expansion of the explants.

[0027] The 1 / 8MS liquid culture medium used in this invention significantly reduces the amount of macroelements required, effectively improving the regeneration rate. Existing research indicates that high concentrations of macroelements reduce plant regeneration induction rates, and excessively high metal ion content inhibits plant differentiation. However, excessively low levels of macroelements can lead to nutrient deficiency and growth stagnation. Furthermore, the lower content of inorganic salts (macroelements) in the culture medium can, to some extent, prevent browning of explants and improve the regeneration rate. See Experimental Example 1 for details.

[0028] 4. This invention employs a sugar-free culture method, promoting the regeneration of *Epipremnum aureum*. Plant tissue culture media typically require the addition of sucrose. Sucrose primarily serves as a carbon and energy source for plant tissues or cells. However, research indicates that *Epipremnum aureum* is a plant tolerant of poor soil conditions and consumes relatively little nutrition and energy. The detached leaves of *Epipremnum aureum* themselves contain a certain amount of sugar that can be broken down and utilized. During cultivation, the detached leaves can synthesize sugars through photosynthesis for their own use. The addition of exogenous sucrose may lead to excessive accumulation of sugars in the culture system, thereby inhibiting regeneration. See Experimental Example 2 for details.

[0029] 5. This invention employs a plant growth regulator-free culture method, significantly improving regeneration efficiency. In tissue culture, plant tissues or cells often lack the ability to synthesize auxin and cytokinin, requiring the addition of exogenous auxin and cytokinin. Under the combined action of these two hormones, the metabolism and balance of endogenous hormones within the tissue and during somatic embryonic development are regulated, completing cell growth, differentiation, and division. It is generally believed that exogenous hormones exert their effects through endogenous hormones. If the type and concentration of exogenous hormones are inappropriate, they will inhibit the activity of endogenous hormones in the explant, thereby preventing organogenesis. The addition of exogenous hormones disrupts the established homeostatic endogenous hormone system, leading to regeneration failure. During the culture process, detached leaves of *Epipremnum aureum* synthesize a certain amount of endogenous hormones (including auxin and cytokinin). These endogenous hormones influence each other, achieving a dynamic balance, thus promoting regeneration. Therefore, this invention employs a plant growth regulator-free culture method. See Experimental Example 3 for details.

[0030] 6. This invention, by adding an appropriate amount of the anti-browning agent PVP, can effectively inhibit browning and improve regeneration. Browning is a common problem in plant tissue culture. Plant tissue browning is caused by the activation of polyphenol oxidase in plant tissues, leading to the accumulation of phenolic compounds into dark macromolecules in cells, resulting in the gradual browning of the culture medium, and ultimately, browning and even death of the explant. Browning is also a common problem in the tissue culture of Araceae ornamental plants. Studies have found that browning is severe in *Epipremnum aureum* tissue culture, requiring the addition of an appropriate amount of anti-browning agent (such as activated carbon). Because this invention uses liquid culture medium, activated carbon is not suitable; therefore, other water-soluble anti-browning agents are selected. Experimental results show that PVP (polyvinylpyrrolidone) is a suitable anti-browning agent for inducing regeneration of detached *Epipremnum aureum* leaves. PVP is an adsorbent for phenolic substances, reducing the degree of plant browning by adsorbing specific types of phenolic compounds. Although phenolic substances exacerbate plant browning, they are also essential for plant growth. PVP does not completely adsorb phenolic substances within the plant, thus allowing for better growth of clustered shoots. See Experiment Example 4 for details.

[0031] 7. Plant tissue culture using liquid culture medium has the following drawbacks: (1) Plant culture requires certain nutrients and growth factors to maintain cell growth and division, and these substances are gradually consumed as time and the number of cells increases. If the culture medium is not changed in time, the concentration of nutrients and growth factors will gradually decrease, eventually leading to cell stagnation or death. (2) Some metabolites and toxic substances, such as excessive phenols, acetic acid, and hydrogen peroxide, will also be produced in the culture medium. If the culture medium is not changed in time, these substances will gradually accumulate and affect cell growth and division. Therefore, the liquid culture medium needs to be changed regularly. However, the more times the liquid culture medium is changed, the better. Too frequent changes will disrupt the established dynamic balance of internal and external metabolism. The results show that under open conditions, it is more appropriate to change the liquid culture medium twice for the regeneration of detached leaves of pothos. See Experimental Example 5 for details.

[0032] 8. This invention uses SPSS24 software for statistical analysis to obtain the suitable conditions for the induction of regeneration of detached leaves of *Epipremnum aureum* under open conditions, resulting in more accurate and reliable results. See Experimental Example 6 for details. Attached Figure Description

[0033] Figure 1 This invention provides a schematic diagram of the leaf state of a hydroponically grown pothos plant after 0 days.

[0034] A is a picture of a green ivy leaf that has been hydroponically grown for 0 days, and B is an enlarged picture of the red box in A.

[0035] Figure 2 This invention provides a schematic diagram of the leaf condition of a green ivy plant after approximately 12 days of hydroponic cultivation.

[0036] Figure 3 This invention provides a schematic diagram of the leaf condition of a green ivy plant after approximately 20 days of hydroponics.

[0037] Figure 4 This invention provides a schematic diagram of the leaf condition of a green ivy plant after approximately 36 days of hydroponic cultivation.

[0038] Figure 5 This invention provides a schematic diagram of the leaf condition of a green ivy plant after approximately 43 days of hydroponic cultivation.

[0039] Figure 6 A schematic diagram of the leaf state of a large-leafed pothos plant grown hydroponically for 0 days according to this invention.

[0040] The image on the right is an enlarged view of the image within the red box on the left.

[0041] Figure 7 A schematic diagram of the state of leaves in a hydroponic large-leaf pothos plant according to this invention;

[0042] The left image shows the hydroponic state after 16 days, and the right image shows the hydroponic state after 38 days.

[0043] Figure 8 Effects of different liquid basal culture media on regeneration induction of detached leaves of *Epipremnum aureum* under open conditions;

[0044] Figure 9 Effects of sucrose on regeneration induction of detached leaves of Epipremnum aureum under open conditions;

[0045] Figure 10 Effects of cytokinins (TDZ and 6-BA) and auxins (IAA and NAA) on regeneration induction of detached leaves of Epipremnum aureum under open conditions;

[0046] Figure 11 Effects of anti-browning agents on regeneration induction of detached leaves of Epipremnum aureum under open conditions;

[0047] Figure 12 The effect of the number of times the liquid basal medium is replaced under open conditions on the induction of regeneration of detached leaves of Epipremnum aureum. Detailed Implementation

[0048] The specific embodiments of the present invention will be further described in detail below with reference to examples.

[0049] Example 1: A method for rapid propagation of the monocotyledonous plant *Epipremnum aureum*

[0050] This embodiment takes the mainstream pothos variety, the green-leaf pothos, as an example, and demonstrates how to propagate green-leaf pothos plants using green-leaf pothos leaves, including the following process steps:

[0051] (1) Cut leaves from the main stem or side branches of the green ivy variety - green ivy, which are 10-40 days old, healthy and free from obvious diseases and pests.

[0052] Leaf cutting method: Use a sharp double-edged blade to quickly cut the leaf from bottom to top, with the cutting point located at the lower part of the petiole (2-3mm away from the stem); the detached pothos leaf does not contain axillary buds and adjacent tissues.

[0053] (2) Treatment of detached pothos leaves, specific methods:

[0054] 1) Cleaning: First, carefully clean the entire leaf with clean tap water 2-3 times, then wipe the entire leaf with medical absorbent cotton moistened with distilled water 2-3 times, and finally clean the entire leaf with distilled water 2-3 times.

[0055] 2) Pruning: Quickly trim the petiole into a wedge shape with a sharp double-edged blade; expose the trimmed petiole to the air in a cool place for 3-4 minutes; try to avoid damaging the leaves throughout the process.

[0056] If the treated leaves cannot be hydroponically grown immediately, wrap the leaves with clean, moist filter paper, wrap the petiole with moist medical absorbent cotton, place them in a seed germination box, and store them in a dark place for 2-3 days.

[0057] (3) Cultivate the treated detached pothos leaves. Specific methods:

[0058] 1) Pour 1 / 4 volume of liquid culture medium into the seed germination box and place a foam board of appropriate size on the surface of the liquid culture medium;

[0059] 2) Place the treated detached leaves of the pothos on a foam board and immerse the petioles in liquid culture medium; place the seed germination box in a clean self-sealing bag, leaving the bag opening unsealed; the culture temperature is 24±2℃, the light intensity is 800lx, and the light duration is 8h / d; during the culture process, the liquid culture medium needs to be changed twice (about once every 20 days).

[0060] The liquid culture medium was 1 / 8 MS liquid medium with added PVP to achieve a PVP concentration of 500 mg / L. The pH of the medium was 6.0, and the composition was: 237.5 mg / L KNO3, 206.25 mg / L NH4NO3, 21.25 mg / L KH2PO4, 46.25 mg / L MgSO4·7H2O, 55 mg / L CaCl2·2H2O, 0.104 mg / L KI, 0.775 mg / L H3BO3, 2.79 mg / L MnSO4·4H2O, 1.075 mg / L ZnSO4·7H2O, 0.03125 mg / L Na2MoO4·2H2O, 0.003125 mg / L CuSO4·5H2O, 0.003125 mg / L CoCl2, and 4.6625 mg / L... Na2·EDTA, 3.475 mg / L FeSO4·7H2O, 12.5 mg / L inositol, 0.25 mg / L glycine, 0.0125 mg / L VB1, 0.0625 mg / L VB6, 0.0625 mg / L VB5.

[0061] (4) After the detached leaves have taken root and sprouted, the detached leaves are removed and planted in soil. They are then managed in a conventional manner, and the soil substrate is garden soil.

[0062] During the cultivation process, it was found that under open conditions, the regeneration process of detached leaves of pothos is roughly as follows: After about 12 days of cultivation, the leaves first form regenerated roots; the regenerated roots continue to develop, and more regenerated roots form at the petiole cut; after about 36 days of cultivation, regenerated buds form at the petiole cut; the regenerated buds continue to develop, forming regenerated seedlings (such as...). Figures 1-5 ).

[0063] The results showed that using the method of this invention, regenerated roots formed in about 12 days of hydroponics, and obvious regenerated buds formed in about 36 days of cultivation. The success rate of regenerated seedlings formed by this method was 90%, and the survival rate of transplanted regenerated plants was close to 100%. This indicates that this method can rapidly and efficiently propagate the mainstream pothos variety, the green-leafed pothos.

[0064] Example 2: A method for rapid propagation of the monocotyledonous plant *Epipremnum aureum*

[0065] This example uses the mainstream pothos variety, large-leaf pothos, to propagate plants using large-leaf pothos leaves. Specific steps are as follows:

[0066] (1) Cut leaves from the main stem or side branches of the large-leaf pothos variety, which are 15-38 days old, healthy and free from obvious diseases and pests.

[0067] Leaf cutting method: Use a sharp double-edged blade to quickly cut the leaf from bottom to top, with the cutting point located at the lower part of the petiole (about 3mm away from the stem); the detached pothos leaf does not include axillary buds and adjacent tissues.

[0068] (2) The specific steps for treating detached pothos leaves are as follows:

[0069] 1) Cleaning: First, carefully clean the entire leaf with clean tap water 2-3 times, then wipe the entire leaf with medical absorbent cotton moistened with distilled water 2-3 times, and finally rinse the entire leaf with distilled water 2-3 times.

[0070] 2) Pruning: Quickly trim the petiole into a wedge shape with a sharp double-edged blade; expose the trimmed petiole to the air in a cool place for about 3 minutes; try to avoid damaging the leaves throughout the process;

[0071] If the treated leaves cannot be hydroponically grown immediately, wrap the leaves with clean, moist filter paper, wrap the petiole with moist medical absorbent cotton, place them in a seed germination box, and store them in a dark place for 3-5 days.

[0072] (3) Cultivate the treated detached pothos leaves. Specific steps:

[0073] 1) Pour 1 / 4 volume of liquid culture medium into the seed germination box and place a foam board of appropriate size on the surface of the liquid culture medium;

[0074] 2) Place the treated detached leaves of the pothos on a foam board and immerse the petioles in liquid culture medium; place the seed germination box in a clean self-sealing bag, leaving the bag opening unsealed; culture temperature 24±2℃, light intensity 800lx, light duration 8h / d; during the culture process, the liquid culture medium needs to be changed twice (about every 20 days).

[0075] The liquid culture medium was prepared by adding PVP to 1 / 8 MS liquid medium to achieve a PVP concentration of 500 mg / L. The pH of the medium was 6.0. The composition of the 1 / 8 MS liquid medium included: 237.5 mg / L KNO3, 206.25 mg / L NH4NO3, 21.25 mg / L KH2PO4, 46.25 mg / L MgSO4·7H2O, 55 mg / L CaCl2·2H2O, 0.104 mg / L KI, 0.775 mg / L H3BO3, 2.79 mg / L MnSO4·4H2O, 1.075 mg / L ZnSO4·7H2O, 0.03125 mg / L Na2MoO4·2H2O, 0.003125 mg / L CuSO4·5H2O, 0.003125 mg / L CoCl2, and 4.6625 mg / L KI. Na2·EDTA, 3.475 mg / L FeSO4·7H2O, 12.5 mg / L inositol, 0.25 mg / L glycine, 0.0125 mg / L VB1, 0.0625 mg / L VB6, 0.0625 mg / L VB5.

[0076] (4) After the detached leaves have taken root and sprouted, the detached leaves are removed and planted in soil. The soil substrate is garden soil, and the plants are managed in a conventional manner.

[0077] Observation of the entire cultivation process revealed that, under open conditions, the regeneration process roughly proceeded as follows: After approximately 16 days of cultivation, regenerated roots first formed on the leaves, and these roots continued to develop, with more regenerated roots forming at the petiole cut; after approximately 38 days of cultivation, regenerated buds formed at the petiole cut; these buds continued to develop, forming regenerated seedlings (see...). Figure 6 , Figure 7 ).

[0078] The results showed that regenerated roots formed in about 16 days of hydroponics, and obvious regenerated buds formed in about 38 days; the success rate of regenerated seedling formation was 88%, and the survival rate of regenerated plants after transplanting was 95%. This indicates that the present invention can rapidly propagate large-leaf pothos.

[0079] In order to obtain a method for rapid propagation of the monocotyledonous plant *Epipremnum aureum*, the applicant conducted extensive experiments, specifically studying the composition of the liquid basal culture medium, as detailed below.

[0080] Experiment Example 1: Screening Experiment of Basic Liquid Culture Media

[0081] Taking the green-leaved pothos as an example, the method of Example 1 was used to set up single-factor experiments with different concentrations of liquid basal culture medium (1, 1 / 2, 1 / 4, 1 / 8, 1 / 10). The liquid basal culture medium did not contain PVP as described in Example 1, and other conditions were the same as in Example 1. Each treatment was repeated 3 times, with 30 leaves each time, and the regeneration rate was counted after 40 days.

[0082] Experiments showed that under open conditions, the regeneration rate of detached leaves of *Epipremnum aureum* initially increased and then decreased as the content of macroelements in the liquid culture medium decreased. The highest regeneration rate, approximately 50%, was achieved when the liquid culture medium concentration was 1 / 8 MS, which was superior to the treatment effect of other concentrations of liquid culture medium. Therefore, 1 / 8 MS is a more suitable liquid culture medium. Results are shown in […]. Figure 8 .

[0083] Experiment Example 2: Effect of sucrose on regeneration-induced pothos

[0084] Taking *Epipremnum aureum* as an example, the method of Example 1 was used to conduct experiments with 1 / 8 MS liquid basal medium containing different concentrations of sucrose. The sucrose concentrations in the medium were 0 g / L, 5 g / L, 10 g / L, 20 g / L, and 30 g / L, respectively. Single-factor experiments were set up. The liquid basal medium did not contain PVP as described in Example 1, and other conditions were the same as in Example 1. Each treatment was repeated 3 times, with 30 leaves each time. The regeneration induction rate was counted after 40 days.

[0085] Experiments showed that under open conditions, the regeneration rate of detached leaves of *Epipremnum aureum* continuously decreased with increasing sucrose content in the liquid culture medium. The highest regeneration rate, approximately 50%, was achieved when the liquid culture medium was 1 / 8 MS and the sucrose concentration was 0 g / L. Other combinations of liquid basal medium and sucrose resulted in lower regeneration rates. Therefore, under open conditions, selecting a liquid basal medium without added sucrose is beneficial for the regeneration of detached leaves of *Epipremnum aureum*. Results are shown below. Figure 9 .

[0086] Experiment Example 3: Effects of Plant Growth Regulators on Regeneration-Induced Pothos

[0087] Taking *Epipremnum aureum* as an example, the method of Example 1 was used to investigate the effects of plant growth regulators on the regeneration induction of detached leaves of *Epipremnum aureum* in 1 / 8 MS liquid basal medium containing different types and concentrations of plant growth regulators. The liquid basal medium did not contain PVP as used in Example 1, and other conditions were the same as in Example 1. The plant growth regulators used were cytokinins (TDZ and 6-BA) and auxins (IAA and NAA). Each treatment was repeated three times, with 30 leaves each time, and the regeneration induction rate was calculated after 40 days.

[0088] (1) Cytokinin TDZ (N-phenyl-N′-1,2,3-thiadiazole-5-urea) was used, and the concentrations of TDZ in the basic liquid culture medium were 0 mg / L, 1 mg / L, 1.5 mg / L, 2 mg / L and 2.5 mg / L, respectively.

[0089] (2) TDZ was replaced with cytokinin 6-BA, and the concentrations of 6-BA in the basal liquid culture medium were 0 mg / L, 0.5 mg / L, 1 mg / L, 1.5 mg / L and 2 mg / L, respectively;

[0090] (3) The auxin IAA was used, and the IAA concentrations in the basic liquid culture medium were 0 mg / L, 0.1 mg / L, 0.2 mg / L, 0.3 mg / L, and 0.4 mg / L, respectively;

[0091] (4) Replace IAA with auxin NAA. The NAA concentrations in the basic liquid culture medium were 0 mg / L, 0.05 mg / L, 0.1 mg / L, 0.2 mg / L and 0.3 mg / L, respectively.

[0092] Experiments showed that under open conditions, treatment with different types and concentrations of cytokinins negatively regulated the regeneration induction of detached *Epipremnum aureum* leaves. The regeneration rate of detached *Epipremnum aureum* leaves continuously decreased with increasing concentrations of TDZ or 6-BA in the liquid basal medium. The highest regeneration rate, approximately 50%, was achieved when the liquid basal medium was 1 / 8 MS and the concentration of TDZ or 6-BA was 0 mg / L. Other combinations of liquid basal medium and cytokinin resulted in significantly lower regeneration rates. Therefore, under open conditions, the absence of cytokinin in the liquid basal medium is beneficial for the regeneration of detached *Epipremnum aureum* leaves.

[0093] Different types and concentrations of auxin all inhibited the induction of regeneration of detached leaves from *Epipremnum aureum*. The regeneration rate of detached leaves continuously decreased with increasing IAA or NAA concentration in the liquid basal medium. The highest regeneration rate (approximately 50%) was achieved when the liquid basal medium was 1 / 8 MS and the IAA or NAA concentration was 0 mg / L. Other combinations of liquid basal medium and auxin resulted in significantly lower regeneration rates. Therefore, the absence of auxin in the liquid basal medium under open conditions is beneficial for the regeneration of detached leaves from *Epipremnum aureum*. (See results below.) Figure 10 .

[0094] Experiment Example 4: Effect of anti-browning agent on regeneration induction of detached leaves of Epipremnum aureum

[0095] Taking the green-leaved pothos as an example, the effect of anti-browning agents on regeneration induction was investigated using the method of Example 1. Different types and concentrations of anti-browning agents were used for treatment, with other conditions the same as in Example 1. Each treatment was repeated 3 times, with 30 leaves each time, and the regeneration induction rate was counted after 40 days.

[0096] The concentrations of the anti-browning agent PVP in the basic liquid culture medium were 0 mg / L, 250 mg / L, 500 mg / L, 1000 mg / L, and 2000 mg / L, and single-factor experiments were set up.

[0097] The anti-browning agent GSH was used to replace PVP in the experiment. The concentrations of GSH in the basic liquid culture medium were 0 mg / L, 10 mg / L, 20 mg / L, 30 mg / L and 40 mg / L, and single-factor experiments were set up.

[0098] Experiments showed that under open conditions, different types of anti-browning agents had varying effects on the regeneration induction of detached leaves of *Epipremnum aureum*. PVP showed better induction effects compared to GSH. With increasing PVP concentration, the regeneration induction rate of detached leaves initially increased and then decreased. The highest regeneration induction rate (60%) was achieved at a PVP concentration of 500 mg / L; the next highest rates (50%, 54.43%, and 53.43%) were achieved at concentrations of 0 mg / L, 250 mg / L, and 1000 mg / L, respectively. The lowest induction rate (45.57%) was observed at a PVP concentration of 2000 mg / L.

[0099] With increasing GSH concentration, the regeneration induction rate also showed a trend of first increasing and then decreasing. At a GSH concentration of 20 mg / L, the highest regeneration induction rate was 55.57%, but this was lower than the highest induction rate (60%) under PVP treatment. Therefore, PVP regeneration induction was more suitable. Figure 11 .

[0100] Experiment Example 5: Test on the Number of Times Liquid Culture Medium Can be Replaced

[0101] Taking the green-leaved pothos as an example, the method of Example 1 was adopted, and the liquid culture medium used the formulation of Example 1 (without PVP as in Example 1). Single-factor experiments were set up with different replacement times (0, 2, 4, 6, 8 times), and other experimental conditions were the same as in Example 1. Each treatment was repeated 3 times, with 30 leaves each time, and the regeneration induction rate was counted after 40 days.

[0102] Experiments showed that under open conditions, the regeneration induction rate of detached leaves of *Epipremnum aureum* initially increased and then decreased with increasing frequency of liquid basal medium replacement. The highest regeneration induction rate (57.8%) was achieved when the liquid basal medium was replaced twice; the regeneration rate was significantly lower for other replacement frequency treatments. Therefore, replacing the liquid basal medium twice is beneficial for the regeneration of detached leaves of *Epipremnum aureum*. Figure 12 .

[0103] Experiment Example 6: Screening of the Optimal Combination for Rapid Propagation of Pothos

[0104] Based on the above single-factor experimental results, the following parameters were used: liquid basal culture medium (1 / 2MS, 1 / 4MS, 1 / 8MS), anti-browning agent (PVP, concentration 250, 500, 1000 mg / L), and the number of liquid culture medium replacements (2, 4, 8 times) according to L9 (3 4An orthogonal experiment was designed. Taking the green-leaved pothos as an example, the method of Example 1 was used to screen suitable conditions for the non-tissue culture regeneration induction of pothos.

[0105] The results (Table 1) show that the A3B2C1 combination exhibited the highest regeneration induction rate for detached leaves of *Epipremnum aureum*, making it a relatively ideal combination. Therefore, under open conditions, the optimal combination of factors for inducing regeneration of detached leaves of *Epipremnum aureum* is: A3B2C1, i.e., 1 / 8 MS liquid basal medium + 500 mg / L PVP + 2 changes of liquid basal medium.

[0106] In addition, referring to Experiments 1-5, the same experiment was conducted on the large-leaved pothos according to the method of Example 2, and the results were consistent with this trend.

[0107] Table 1. Homogeneous subsets comparing the regeneration rates of detached leaves from different combinations (T) of pothos.

[0108]

[0109] Note: T: Combination; A1: 1 / 2 liquid basal medium, A2: 1 / 4 liquid basal medium, A3: 1 / 8 liquid basal medium; B1: 250 mg / L PVP, B2: 500 mg / L PVP, B3: 1000 mg / L PVP; C1: liquid basal medium changed 2 times, C2: liquid basal medium changed 4 times, C3: liquid basal medium changed 8 times.

Claims

1. A method for rapidly propagating the monocotyledonous plant *Epipremnum aureum*, characterized in that: Includes the following steps: (1) Cut healthy, disease-free leaves from the main stem or side branches of the mother plant, which are 10-40 days old, and are free from obvious pests and diseases. (2) Processing detached pothos leaves: After washing, trim the petioles and cut them into wedge shapes; (3) Cultivate the treated detached pothos leaves: Place the leaf surface on a foam board and immerse the petiole in liquid culture medium for cultivation; (4) After the detached leaves have taken root and sprouted, remove the detached leaves and cultivate them in soil, and manage them in a routine manner; The method for cutting the leaves of the pothos described in step (1) is as follows: use a sharp double-edged blade to quickly cut the leaves from bottom to top, with the cutting point located at the lower part of the petiole and 2-3 mm away from the stem; the detached pothos leaves do not contain axillary buds or adjacent tissues; The liquid culture medium in step (3) is as follows: PVP is added to 1 / 8MS basal liquid culture medium. The PVP concentration is 500 mg / L, and the pH is 6.

0. The specific composition is: 237.5 mg / L KNO3, 206.25 mg / L NH4NO3, 21.25 mg / L KH2PO4, 46.25 mg / L MgSO4•7H2O, 55 mg / L CaCl2•2H2O, 0.104 mg / L KI, 0.775 mg / L H3BO3, 2.79 mg / L MnSO4•4H2O, 1.075 mg / L ZnSO4•7H2O, 0.03125 mg / L Na2MoO4•2H2O, 0.003125 mg / L CuSO4•5H2O, 0.003125 mg / L CoCl2, 4.6625 mg / L MgSO4•7H2O, 0.003125 mg / L CuSO4•5H2O, 0.003125 mg / L CoCl2, 4.6625 mg / L MgSO4•7H2O, 0.003125 mg / L CuSO4•5H2O, 0.003125 mg / L CoCl2, 0.0031 ...7H2O, 0.003125 mg / L CuSO4•7H2O, 0.003125 mg / L CuSO4•7 Na2•EDTA•2H2O, 3.475 mg / L FeSO4•7H2O, 12.5 mg / L inositol, 0.25 mg / L glycine, 0.0125 mg / L VB1, 0.0625 mg / L VB6, 0.0625 mg / L VB5; During the in vitro cultivation of pothos leaves, the liquid culture medium was changed twice, once every 20 days.

2. The method according to claim 1, characterized in that: The specific steps for processing the detached pothos leaves are as follows: 1) Cleaning: First, carefully clean the entire leaf 2-3 times with clean tap water, then wipe the entire leaf 2-3 times with medical absorbent cotton moistened with distilled water, and finally clean the entire leaf 2-3 times with distilled water. 2) Pruning: Use a sharp double-edged blade to quickly cut the petiole into a wedge shape. Expose the pruned petiole to the air in a shady place for 3-4 minutes. Avoid damaging the leaves throughout the process.

3. The method according to claim 1, characterized in that: If the leaves treated in step (2) cannot be hydroponically grown immediately, wrap the leaves with clean, moist filter paper, wrap the petiole with moist medical absorbent cotton, place them in a seed germination box, and store them in a dark place for later use.

4. The method according to claim 1, characterized in that: The specific steps for culturing the treated detached pothos leaves are as follows: 1) Pour 1 / 4 volume of liquid culture medium into the seed germination box and place a foam board on the surface of the liquid culture medium; 2) Place the treated detached leaves of the pothos on a foam board and immerse the petioles in liquid culture medium; place the seed germination box in a clean self-sealing bag, leaving the bag open; the culture temperature is 24±2 ℃, the light intensity is 800 lx, and the light duration is 8 h / d.

5. The method according to claim 1, characterized in that: When growing detached leaves in soil, the substrate is garden soil.

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