Sugar-free tissue culture method of fissurea villosa

Abstract

The invention relates to the technical field of plant culture, and in particular relates to a sugar-free tissue culture method of fissuria viridulosa, which comprises the following steps: (1) preparing a plant material; (2) preparing a sugar-free culture medium; (3) inoculating: inoculating the strong seedlings screened in the step (1) into a sugar-free culture square box; (4) sugar-free culture: placing the inoculated culture square box in a culture room, and culturing under an illumination condition; (5) transplanting and maintaining: cleaning a root culture medium of the cultured seedlings, planting the seedlings in peat soil, placing the seedlings in a greenhouse, and irrigating a water-soluble fertilizer once a week after transplanting and rooting. According to the method, CO2 is adopted to replace cane sugar to serve as the only carbon source in the culture medium, so that the tissue culture seedlings are converted from heterotrophy to autotrophy, energy is generated through photosynthesis to be supplied to self growth, and the risk of microbial contamination is fundamentally reduced. Experimental data show that the pollution rate of the method is remarkably lower than that of a traditional culture method.

Description

Technical Field

[0001] This invention relates to the field of plant culture technology, specifically to a sugar-free tissue culture method for Philodendron pinnatifida. Background Technology

[0002] Philodendron selloum is a foliage plant with high ornamental value, and its propagation mainly relies on tissue culture technology. Currently, sugar is commonly used as a carbon source to provide energy for tissue culture seedlings. Therefore, contamination is easily caused during the production and transportation of tissue culture seedlings, especially during export transportation. If contamination occurs, the entire batch of seedlings may be destroyed and lost. Furthermore, heterotrophic growth in a closed environment results in weaker seedling quality, and transplanting outdoors can lead to low survival rates due to the seedlings' inability to adapt to the external environment. In addition, the relatively high humidity and low CO2 concentration inside the tissue culture bottles and containers, combined with the use of sugar as a carbon source for heterotrophic growth, leads to poor photosynthetic capacity, poor growth and rooting quality, and also makes the seedlings susceptible to microbial contamination.

[0003] Sugar-free tissue culture, a novel plant micropropagation technique, uses CO2 instead of sugar as the sole carbon source for plants, transforming tissue-cultured seedlings from a commensal to an autotrophic species, thus producing high-quality seedlings. This technique avoids microbial contamination to a certain extent, improves transplant survival rates, and eliminates the need for hardening-off processes, allowing direct planting in seedling cups. Currently, sugar-free tissue culture has been applied to various crops such as apples, sweet potatoes, tea trees, chrysanthemums, and bananas, but there are no reports on sugar-free tissue culture of Philodendron.

[0004] To address the aforementioned issues, there is an urgent need to develop an efficient sugar-free tissue culture rooting system for Philodendron to reduce contamination rates, improve seedling quality and transplant survival rates, and facilitate industrialized seedling production. Summary of the Invention

[0005] To address the problems existing in the prior art, the purpose of this invention is to provide a sugar-free tissue culture method for Philodendron pinnatifida.

[0006] The objective of this invention is achieved through the following technical solution:

[0007] A sugar-free tissue culture method for *Philodendron pinnatifida* includes the following steps:

[0008] (1) Preparation of plant materials: The short stems of Philodendron pinnatifidum were used as explants. After disinfection, they were inoculated into induction medium, proliferation medium and seedling strengthening medium in sequence. Sterile seedlings were screened.

[0009] (2) Preparation of sugar-free culture medium: MS stock solution without organic components is used as the base culture medium. 0.5 mg / L of indolebutyric acid (I6-benzylaminopurine (6-BA)) and a solidifying agent are added to adjust the pH to 6.0-6.2. No exogenous sugar is added to obtain the sugar-free culture medium, which is then dispensed into sugar-free culture boxes.

[0010] (3) Inoculation: Inoculate the strong seedlings selected in step (1) into sugar-free culture boxes;

[0011] (4) Sugar-free culture: Place the inoculated culture box in the culture room and culture under light conditions;

[0012] (5) Transplanting and maintenance: After the seedlings have been cultured, clean the culture medium from the roots, plant them in peat soil, place them in a greenhouse, and water them with water-soluble fertilizer once a week after they have taken root.

[0013] Preferably, in step (1), the induction medium is MS + 6-benzylaminopurine (6-BA) 3.0 mg / L + naphthaleneacetic acid (NAA) 0.01 mg / L + sucrose 30 g / L + carrageenan 6.0 g / L; the proliferation medium is MS + 6-benzylaminopurine (6-BA) 2.0 mg / L + naphthaleneacetic acid (NAA) 0.1 mg / L + sucrose 30 g / L + carrageenan 6.0 g / L; and the seedling strengthening medium is MS + activated carbon 0.2 g / L + sucrose 30 g / L + carrageenan 6.0 g / L.

[0014] Preferably, in step (1), the explant disinfection process is as follows: soak in 75% ethanol for 30 seconds, then soak in 0.1% mercuric chloride for 8-10 minutes, and then rinse with sterile water 3-5 times.

[0015] Preferably, in step (1), the sterile seedlings are cultured seedlings with a height of 3.0-3.5cm and "three leaves and one heart".

[0016] Preferably, in step (2), the coagulant is one of carrageenan, agar or gellan gum; the concentration of the coagulant is: 6.0 g / L carrageenan, 5.5 g / L agar or 4.0 g / L gellan gum.

[0017] Preferably, in step (2), the sugar-free culture medium is sterilized at 121°C and 0.11MPa for 20 minutes and then dispensed.

[0018] Preferably, in step (3), the sugar-free culture box has a built-in acupuncture tray and the top sealing film has ventilation holes.

[0019] Preferably, in step (4), the temperature in the culture room is controlled at 25°C, the photoperiod is 12h / day, an LED light source is used, the light intensity of the LED light source is 5000lx, CO2 is naturally introduced through a CO2 gas tank during the light period, and the CO2 is stopped during the dark period, and the culture lasts for 30 days.

[0020] Preferably, in step (4), the CO2 concentration in the culture chamber during the light period is maintained at 800-1200 ppm.

[0021] Preferably, in step (5), the temperature of the greenhouse is 18-32℃, the humidity is 60-80%, and the light intensity is 6000-10000lx.

[0022] The beneficial effects of this invention are as follows:

[0023] 1. This invention uses CO2 instead of sucrose as the sole carbon source in the culture medium, transforming tissue culture seedlings from heterotrophic to autotrophic. The seedlings then generate energy through photosynthesis to support their growth, fundamentally reducing the risk of microbial contamination. Experimental data show that the contamination rate using this method is significantly lower than that of traditional culture methods.

[0024] 2. Commercially available sugar-free experiments typically use perlite or vermiculite as a support, but this invention uses a gel, ensuring that the cultured seedlings can be directly planted in soil. By optimizing the culture medium formula, the permeability of the culture container, and the parameters of the culture environment, this invention significantly improves the rooting rate and root quality of Philodendron tissue culture seedlings.

[0025] 3. The tissue culture seedlings cultivated by the method of this invention have better growth indicators such as plant height, stem diameter, leaf length and leaf width than those of traditional cultivation methods. The plants are vigorous and the leaves are bright green.

[0026] 4. Sugar-free tissue culture seedlings cultivated using the method of this invention do not require a hardening-off step and can be directly transplanted, which simplifies the production process and improves the transplant survival rate.

[0027] 5. The process of this invention is simple, requires no addition of sucrose, reduces the seedling screening steps, helps to reduce the cost of factory seedling production, and facilitates the promotion and application of new technologies.

[0028] 6. The method of this invention is applicable to a variety of Philodendron varieties, such as Angel of the Upright Leaf, Little Angel, and Hope, and has broad application prospects. Attached Figure Description

[0029] The present invention will be further described with reference to the accompanying drawings, but the embodiments in the drawings do not constitute any limitation on the present invention. For those skilled in the art, other drawings can be obtained based on the following drawings without creative effort.

[0030] Figure 1This is a comparative diagram of the transplantation of sugar-free cultured seedlings (treatment group) and traditional tissue culture seedlings (control group) in Experiment Example 4. Detailed Implementation

[0031] The technical solution of the present invention is illustrated below through specific examples. It should be understood that the one or more method steps mentioned in the present invention do not preclude the existence of other method steps before or after the combined steps, or the insertion of other method steps between these explicitly mentioned steps; it should also be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the present invention. Furthermore, unless otherwise stated, the numbering of each method step is merely a convenient tool for identifying each method step, and not for limiting the order of the method steps or defining the scope of the present invention. Changes or adjustments to their relative relationships, without substantially altering the technical content, should also be considered within the scope of the present invention.

[0032] To better understand the above technical solutions, exemplary embodiments of the present invention are described in more detail below. While exemplary embodiments of the present invention are shown, it should be understood that the present invention can be implemented in various forms and should not be limited to the embodiments set forth herein. Rather, these embodiments are provided to enable a more thorough understanding of the present invention and to fully convey the scope of the invention to those skilled in the art.

[0033] The present invention will be further described below with reference to the following embodiments.

[0034] Example

[0035] A sugar-free tissue culture method for *Philodendron pinnatifida* includes the following steps:

[0036] Step 1: Preparation of plant materials

[0037] Aseptic tissue culture seedlings of Philodendron simsii were selected as the culture material, and the preparation process is as follows:

[0038] (1) Explant treatment: The short stems of Philodendron simsii were used as explants. After surface disinfection (conventional tissue culture disinfection procedure, such as soaking in 75% ethanol for 30s + soaking in 0.1% mercuric chloride for 8-10min), they were ready for use.

[0039] (2) Induction culture: The sterilized explants were inoculated into the induction culture medium, which was formulated as follows: MS basic medium + 6-benzylaminopurine (6-BA) 3.0 mg / L + naphthaleneacetic acid (NAA) 0.01 mg / L + sucrose 30 g / L + carrageenan 6.0 g / L, pH 5.8-6.0, and cultured until adventitious shoots were induced;

[0040] (3) Proliferation culture: The adventitious shoots were transferred to the proliferation medium, which was formulated as follows: MS basic medium + 6-benzylaminopurine (6-BA) 2.0 mg / L + naphthaleneacetic acid (NAA) 0.1 mg / L + sucrose 30 g / L + carrageenan 6.0 g / L, pH 5.8-6.0, for large-scale propagation;

[0041] (4) Seedling cultivation: When the number of propagated seedlings reaches a certain level, they are transferred to seedling cultivation medium with the following formula: MS basic medium + activated carbon 0.2 g / L + sucrose 30 g / L + carrageenan 6.0 g / L, pH 5.8-6.0, and cultured until the shoots elongate;

[0042] (5) Material selection: Select robust seedlings with a height of 3.0-3.5cm and "three leaves and one heart" as the initial material for sugar-free culture.

[0043] Step 2. Preparation of sugar-free culture medium

[0044] (1) The sugar-free culture medium formula is: MS basic culture medium (stock solution without organic components) + indolebutyric acid (I6-benzylaminopurine (6-BA)) 0.5 mg / L + solidifying agent, pH adjusted to 6.0-6.2, without adding any exogenous sugars (such as sucrose, glucose).

[0045] The coagulant can be any of the following: carrageenan: 6.0 g / L; agar: 5.5 g / L; gellan gum: 4.0 g / L.

[0046] (2) After sterilizing the prepared culture medium at 121℃ and 0.11MPa for 20 minutes, dispense it into sugar-free culture containers and let it cool and solidify for later use.

[0047] Step 3. Selection of culture container

[0048] The disposable sugar-free culture box measures 9cm×9cm×10cm and integrates a 16-well tray (each well corresponds to one seedling). The sealing film at the top of the box has a ventilation hole (0.5-1cm in diameter) for gas exchange.

[0049] Step 4. Inoculation Method

[0050] The selected robust seedlings were inoculated one by one into the plug trays of the sugar-free culture box, with 16 seedlings inoculated in each box, and 10 replicate boxes were set up for each treatment (i.e., the total inoculation amount per treatment was 160 seedlings).

[0051] Step 5. Sugar-free culture conditions

[0052] (1) Environmental parameters: The temperature of the culture room is controlled at 25℃ and the photoperiod is 12h / day (12h during the light period and 12h during the dark period).

[0053] (2) Light source: LED lights are used as the light source, and the light intensity is controlled at 5000 lx (this intensity is the optimal light condition, which can avoid the excessive growth caused by low light or the withered leaves caused by high light).

[0054] (3) CO2 supply: CO2 cylinders are used as the CO2 source and CO2 is supplied through natural input; CO2 is released only during the light cycle to maintain the CO2 concentration in the culture room at 800-1200ppm; CO2 release is stopped during the dark cycle;

[0055] (4) Culture cycle: 30 days of continuous culture.

[0056] Step 6. Transplanting and Post-Transplant Care

[0057] (1) Pre-transplanting treatment: Take out the sugar-free tissue culture seedlings that have been cultured for 30 days, and clean the roots with sterile water to remove the residual culture medium, so as to avoid contamination after transplanting caused by the residual culture medium;

[0058] (2) Cultivation substrate: Peat moss is used as the transplanting substrate (pH 5.5-6.5, organic matter content ≥30%). The planting container can be a 128 mesh seedling tray or a 90-mesh flower pot.

[0059] (3) Greenhouse maintenance conditions: After transplanting, place the seedlings in a simple greenhouse, with the temperature controlled at 18-32℃, the air humidity controlled at 60-80%, and the light intensity controlled at 6000-10000lx;

[0060] (4) Water and fertilizer management: After transplanting and rooting, water once a week with a 2000-fold diluted 20-20-20 balanced water-soluble fertilizer (N:P2O5:K2O=20:20:20) to supplement nutrients.

[0061] The composition of MS basal medium is shown in the table below:

[0062]

[0063] Experiment Example 1: Effect of different light intensities on sugar-free culture

[0064] 1. Experimental Design

[0065] Treatment groups: Three light intensity gradients were set up, namely 3000 lx (treatment 1), 5000 lx (treatment 2), and 8000 lx (treatment 3). All groups used the sugar-free culture medium (MS without organic components + indolebutyric acid (I6-benzylaminopurine (6-BA)) 0.5 mg / L + carrageenan 5.5 g / L) and culture conditions (CO2 800-1200 ppm, 25℃, 12h photoperiod).

[0066] Control group: Traditional saccharified tissue culture, culture medium was MS + indolebutyric acid (I6-benzylaminopurine (6-BA)) 0.5 mg / L + sucrose 30 g / L + carrageenan 5.5 g / L, light intensity 3000 lx, other conditions were the same as the treatment group;

[0067] Replication and cycle: 10 boxes per treatment, 16 plants per box, cultured for 30 days, and the contamination rate, rooting rate, plant height, number of leaves, root length and plant condition were recorded.

[0068] 2. Experimental Results

[0069] Treatment Irradiance 30-day contamination rate 30-day rooting rate Plant height (cm) Leaf number Root length (cm) Plant status Control 3000lx 30.00% 100.00% 3.9 3.9 4.4 Seedling short No sugar treatment 1 3000lx 0.00% 100.00% 4.9 4.1 5.1 Seedling thin, overgrowth No sugar treatment 2 5000lx 0.00% 100.00% 4.5 5.1 5.5 Seedling strong, dark green leaf No sugar treatment 3 8000lx 0.00% 100.00% 3.8 4.8 3.9 Seedling dry tip, yellow leaf

[0070] 3. Conclusion

[0071] 5000 lx is the optimal light intensity for sugar-free culture, which can balance plant height, number of leaves and root development, and avoid excessive growth or leaf dieback.

[0072] Experiment Example 2: Effect of different CO2 concentrations on the effect of sugar-free culture

[0073] 1. Experimental Design

[0074] Treatment groups: Two CO2 concentration gradients were set up, namely 800-1200ppm (treatment 1) and 1200-1500ppm (treatment 2), both using the sugar-free culture medium and culture conditions of the present invention (light intensity 5000lx, 25℃, 12h photoperiod).

[0075] Control group: Traditional saccharified tissue culture, CO2 concentration at ambient level (approximately 400 ppm), other conditions the same as treatment group;

[0076] Replication and cycle: 10 boxes were treated per treatment, 16 plants per box, and cultured for 30 days. Relevant indicators were then statistically analyzed.

[0077] 2. Experimental Results

[0078] Treatment [CO2 concentration] 30-day contamination rate 30-day rooting rate Plant height (cm) Leaf number Root length (cm) Plant status Control ~400 ppm 20.00% 100.00% 3.9 3.9 4.4 Seedling short No sugar treatment 1 800-1200 ppm 0.00% 100.00% 4.5 5.1 5.5 Seedling strong, dark green leaf No sugar treatment 2 1200-1500 ppm 0.00% 100.00% 4.3 5.2 5.0 Seedling strong, dark green leaf

[0079] 3. Conclusion

[0080] 800-1200 ppm is the optimal CO2 concentration for sugar-free culture, at which plant height and root length are better than those treated with higher CO2 concentrations.

[0081] Experiment Example 3: Effects of Different Coagulants on Sugar-Free Culture

[0082] 1. Experimental Design

[0083] Treatment groups: Three coagulants were set up: carrageenan (6.0 g / L, treatment 1), agar (5.5 g / L, treatment 2), and gellan gum (4.0 g / L, treatment 3). All three groups were cultured using the sugar-free culture medium and conditions of this invention (5000 lx light, 800-1200 ppm CO2).

[0084] Replication and cycle: 10 boxes were treated per treatment, 16 plants per box, and cultured for 30 days. Relevant indicators were then statistically analyzed.

[0085] 2. Experimental Results

[0086] Treatment Coagulant 30-day rooting rate Plant height (cm) Leaf number Root length (cm) Plant status Treatment 1 Carrageenan 100.00% 4.6 4.9 5.2 Seedling strong, large leaf Treatment 2 Agar 100.00% 4.4 5.0 4.9 Seedling strong, large leaf Treatment 3 Gellan gum 100.00% 4.5 4.7 5.1 Seedling strong, large leaf

[0087] 3. Conclusion

[0088] Carrageenan, agar, and gellan gum can all be used as solidifying agents for sugar-free culture media, with no significant difference in effectiveness. The choice can be made based on cost and ease of operation.

[0089] Experiment Example 4: Comparison of Transplantation of Sugar-Free Cultured Seedlings and Traditional Tissue Cultured Seedlings

[0090] 1. Experimental Design

[0091] Treatment group: Seedlings cultured without sugar for 30 days according to the present invention were transplanted into 128-mesh plug trays and 90-mesh flower pots respectively;

[0092] Control group: Seedlings cultured in traditional sugar-containing tissue culture for 30 days, with the same transplanting containers and subsequent maintenance conditions as the treatment group;

[0093] Repetition and cycle: 80 plants were planted in each container type and kept in a greenhouse for 2 months. The survival rate and growth indicators were then recorded.

[0094] 2. Experimental Results

[0095] (1) Transplantation with 128-mesh seedling tray (1 month)

[0096] Treatment Survival rate Plant height (cm) Crown width (cm) Leaf number Leaf length (cm) Leaf width (cm) Control 93.06% 5.1 6.2 4.8 2.4 3.4 Treatment group 100.00% 6.6 7.3 6.1 3.3 4.5

[0097] (2) Transplanting in 90mm flower pots (2 months)

[0098] Treatment Survival rate Plant height (cm) Crown width (cm) Leaf number Leaf length (cm) Leaf width (cm) Control 81.94% 6.0 7.1 5.3 3.2 4.6 Treatment group 100.00% 7.1 8.3 6.9 3.8 5.2

[0099] 3. Conclusion

[0100] The transplant survival rate, plant height, crown width, and number of leaves of sugar-free cultured seedlings were significantly better than those of traditional tissue culture seedlings, demonstrating excellent transplanting results. Furthermore, the planting conditions of the treatment and control groups were as follows: Figure 1 As shown.

[0101] The above tests show that the sugar-free tissue culture method of the present invention has the following significant advantages compared with traditional saccharified tissue culture:

[0102] (1) The contamination rate is significantly reduced: the contamination rate of traditional tissue culture is 20-30% after 30 days, while the contamination rate of sugar-free culture of this invention is 0% after 30 days, which completely solves the problem of contamination and disposal during export transportation;

[0103] (2) Seedling quality has been greatly improved:

[0104] Plant height: Sugar-free culture seedlings can reach a height of 4.5cm (compared to 3.9cm for traditional tissue culture seedlings), an increase of 15.38%;

[0105] Leaf count: Sugar-free culture seedlings can have up to 5.1 leaves (compared to 3.9 leaves for traditional tissue culture seedlings), an increase of 30.77%;

[0106] Root length: The root length of sugar-free culture seedlings can reach 5.5cm (compared to 4.4cm for traditional tissue culture seedlings), an increase of 25.00%;

[0107] Moreover, the seedlings showed no excessive growth or withered leaves, the plants were robust with dark green leaves, and their self-sufficiency was fully activated;

[0108] (3) Excellent transplant survival rate and growth performance:

[0109] Transplanting in 128-mesh seedling trays: 100% survival rate for sugar-free seedlings (93.06% for traditional seedlings), plant height 6.6cm (5.1cm for traditional seedlings), an increase of 29.41%;

[0110] Transplanting in 90mm pots: Sugar-free seedlings had a 100% survival rate (compared to 81.94% for traditional seedlings), and a plant height of 7.1cm (compared to 6.0cm for traditional seedlings), representing an increase of 18.33%.

[0111] In addition, sugar-free seedlings recover quickly after transplanting (the recovery period is shortened by 30%), have strong resistance (improved resistance to low temperature and drought), and grow rapidly (crown width, leaf length, and leaf width all increase by more than 13%).

[0112] In the description of this specification, the references to terms such as "one embodiment," "some embodiments," "example," "specific example," or "some examples," etc., indicate that a specific feature, structure, material, or characteristic described in connection with that embodiment or example is included in at least one embodiment or example of the invention. The illustrative expressions of the above terms in this specification should not be construed as necessarily referring to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples. In addition, those skilled in the art can combine and integrate the different embodiments or examples described in this specification.

[0113] Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention. Those skilled in the art can make changes, modifications, substitutions and variations to the above embodiments within the scope of the present invention.

Claims

1. A sugar-free tissue culture method for *Philodendron pinnatifida*, characterized in that, Includes the following steps: (1) Preparation of plant materials: The short stems of Philodendron pinnatifidum were used as explants. After disinfection, they were inoculated into induction medium, proliferation medium and seedling strengthening medium in sequence. Sterile seedlings were screened. (2) Preparation of sugar-free culture medium: MS with no organic components as the base culture medium, indolebutyric acid and coagulant are added, pH is adjusted to 6.0-6.2, no exogenous sugar is added, sugar-free culture medium is obtained and dispensed into sugar-free culture boxes; (3) Inoculation: Inoculate the strong seedlings selected in step (1) into sugar-free culture boxes; (4) Sugar-free culture: Place the inoculated culture box in the culture room and culture under light conditions; (5) Transplanting and maintenance: Plant the cultivated seedlings in peat soil and place them in a greenhouse. After the seedlings have taken root, water them with water-soluble fertilizer once a week.

2. The method for sugar-free tissue culture of *Philodendron pinnatifida* according to claim 1, characterized in that, In step (1), the induction medium is MS + 6-benzylaminopurine 3.0 mg / L + naphthaleneacetic acid (NAA) 0.01 mg / L + sucrose 30 g / L + carrageenan 6.0 g / L; the proliferation medium is MS + 6-benzylaminopurine 2.0 mg / L + naphthaleneacetic acid (NAA) 0.1 mg / L + sucrose 30 g / L + carrageenan 6.0 g / L; and the seedling strengthening medium is MS + activated carbon 0.2 g / L + sucrose 30 g / L + carrageenan 6.0 g / L.

3. The method for sugar-free tissue culture of *Philodendron pinnatifida* according to claim 1, characterized in that, In step (1), the explant disinfection process is as follows: soak in 75% ethanol for 30 seconds, then soak in 0.1% mercuric chloride for 8-10 minutes, and then rinse with sterile water 3-5 times.

4. The method for sugar-free tissue culture of *Philodendron pinnatifida* according to claim 1, characterized in that, In step (1), the sterile seedlings are cultured seedlings with a height of 3.0-3.5cm and "three leaves and one heart".

5. The method for sugar-free tissue culture of *Philodendron pinnatifida* according to claim 1, characterized in that, In step (2), the coagulant is one of carrageenan, agar or gellan gum; the concentration of the coagulant is: 6.0 g / L carrageenan, 5.5 g / L agar or 4.0 g / L gellan gum.

6. The method for sugar-free tissue culture of *Philodendron pinnatifida* according to claim 1, characterized in that, In step (2), the sugar-free culture medium is sterilized at 121℃ and 0.11MPa for 20 minutes and then dispensed.

7. The method for sugar-free tissue culture of *Philodendron pinnatifida* according to claim 1, characterized in that, In step (3), the sugar-free culture box has a built-in acupuncture tray and a vent hole in the top sealing film.

8. The method for sugar-free tissue culture of *Philodendron pinnatifida* according to claim 1, characterized in that, In step (4), the temperature in the culture room is controlled at 25°C, the photoperiod is 12h / day, an LED light source is used, the light intensity of the LED light source is 5000lx, CO2 is naturally introduced through a CO2 gas tank during the light period, and the CO2 is stopped during the dark period, and the culture lasts for 30 days.

9. The method for sugar-free tissue culture of *Philodendron pinnatifida* according to claim 1, characterized in that, In step (4), the CO2 concentration in the culture chamber during the light period is maintained at 800-1200 ppm.

10. A method for sugar-free tissue culture of *Philodendron pinnatifida* according to claim 1, characterized in that, In step (5), the temperature of the greenhouse is 18-32℃, the humidity is 60-80%, and the light intensity is 6000-10000lx.

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