A method for cultivating seedlings of elaeagnus mollis by sugar-free tissue culture in light environment

Abstract

The application provides a kind of light environment sugar-free tissue culture seedling raising method of Cervus grass leaf, uses light autotrophy sugar-free tissue culture method, for the development stage of whole leaf's rooting and strong seedling rooting promotion, a set of light environment cultivation method is researched, i.e., the substrate for seedling raising is mixed with peat and perlite according to the volume ratio of 5:1, the whole dark culture is used for 10d in leaf rooting induction period, 1000lx weak light and 12h / d photoperiod culture is used for 2 months, strong seedling rooting period adopts 3000lx strong light, 12h / d photoperiod culture is used for 20d, the whole cycle is matched with intermittent CO2 common giving and temperature is kept at 27 DEG C culture, the technical problems of long traditional Cervus grass seedling raising cycle, high pollution rate and low transplanting survival rate are solved, which can provide technical basis for large-scale, factory seedling breeding of Cervus grass.

Description

Technical Field

[0001] This invention belongs to the field of plant seedling cultivation, specifically relating to a method for sugar-free tissue culture of *Deer Antler Grass* leaves under light conditions. Background Technology

[0002] Deer antler grass is a perennial herbaceous medicinal plant belonging to the genus Deer antler grass of the family Scrophulariaceae. It has clear medicinal value and market demand for cultivation. The large-scale and standardized supply of high-quality seedlings has been the focus of the development of the artificial cultivation industry of deer antler grass in recent years. Currently, the seedling cultivation technology of *Deer Antler Grass* mainly includes two categories: field sowing seedlings and conventional sugar-containing tissue culture seedlings. Meanwhile, photoautotrophic sugar-free tissue culture technology, as a general tissue culture technology, has been studied and applied in various plants such as *Pinellia ternata* (Zhan Yan et al., 2009) and potato (Fan Yi et al., 2025). However, through our attempts to apply it to *Deer Antler Grass*, we found that there are still many problems. Existing sugar-free tissue culture for crops such as sweet potato uses pre-differentiated tissue culture seedlings and budded stem segments as starting materials, and is only used in the strong seedling rooting stage. There is no sugar-free tissue culture rapid propagation system specifically for *Deer Antler Grass* leaves, and general sugar-free tissue culture parameters cannot solve the problems of low rooting rate of *Deer Antler Grass* leaves and the delicate root system being prone to root rot. For example, existing *Deer Antler Grass* tissue culture seedlings mostly use stem segments as explants, and when using sugar-containing culture media with added sucrose for closed culture, the carbon source for the plant is supplied through heterotrophic means. This technology requires the cultivation of adventitious buds followed by root development. The addition of sucrose to the culture medium easily breeds miscellaneous bacteria, resulting in a high contamination rate during the cultivation process. Under heterotrophic conditions, seedling photosynthetic tissue development is weak, leading to a long recovery period after transplanting, low survival rates, and an overall longer seedling cycle, thus increasing the comprehensive cost for large-scale production. While plant photoautotrophic sugar-free tissue culture technology uses exogenous CO2 to replace sucrose as a carbon source, inducing autonomous photosynthetic growth, this technology, when applied to *Deer Antler Grass*, cannot provide a method for directly inducing rooting in a sugar-free system using leaves as explants. Although *Deer Antler Grass* leaves are readily available, their differentiation conditions are demanding, and existing methods often result in low rooting rates and poor development. Furthermore, we found that existing sugar-free tissue cultures mostly use constant light intensity and do not select appropriate light intensity and time parameters for different developmental stages of *Deer Antler Grass* leaf-induced rooting and seedling vigorous rooting. This easily causes photosynthetic stress and makes it impossible to achieve efficient seedling growth in a short period. The existing CO2 gas supply concentration range is quite large, basically ranging from 600ppm to 10000ppm, and most of them use continuous high-concentration gas supply or dark period gas supply. There is no low-concentration gas supply specifically suitable for the photosynthetic characteristics of *Deer Antler Grass* for regulation. This easily leads to carbon source waste and photosynthetic inactivation of plants, making it difficult to achieve optimal photoautotrophic efficiency at low cost.

[0003] In summary, current tissue culture techniques for *Deer Antler Grass* cannot simultaneously shorten the seedling cycle and reduce production costs while improving seedling transplant survival rates. Therefore, it is necessary to develop a photoautotrophic sugar-free tissue culture method specifically for *Deer Antler Grass*. Summary of the Invention

[0004] To address the shortcomings of existing technologies, this invention aims to solve the problems of long seedling growth cycles, trait separation, and numerous pests and diseases associated with traditional sowing methods, as well as high contamination rates, high costs, and low transplant survival rates associated with conventional sugar-containing tissue culture. The invention improves upon general sugar-free tissue culture technology, adapting it for seedling propagation of *Deer Antler Grass* by directly inducing root growth from leaves, shortening the growth cycle, increasing seedling speed, and achieving short-cycle, low-cost, and high-quality large-scale seedling propagation of *Deer Antler Grass*.

[0005] To address the aforementioned problems, this application provides a method for sugar-free tissue culture of *Deer Antler Grass* leaves under light conditions, specifically including the following steps: Prepare the sugar-free matrix: Mix peat and perlite at a volume ratio of 5:1; the peat can be 0-10mm Pints ​​peat. Disinfection pretreatment: Prepare a 0.1% potassium permanganate solution, and completely immerse the sugar-free matrix from step (1) in the potassium permanganate solution for 4 hours; (3) Leaf explant inoculation and dark culture: Select disease-free, mature and complete leaves of *Deer Antler Grass* and inoculate them into the sterilized sugar-free substrate of step (2); place them in a sugar-free incubator and continue dark culture for 10 days. This stage mainly causes the base of the leaves to scab. (4) Staged light cultivation: The first stage of low-light cultivation: After the dark cultivation is completed, turn on the light and control the light intensity to 1000 lx, with 12 hours of light per day, and cultivate continuously for 2 months to promote root development. The second stage is the cultivation of strong seedlings under strong light: After the seedlings reach a height of 3cm, the light intensity is increased to 3000lx, with 12 hours of light per day, and this is continued for 20 days to strengthen the photosynthetic tissue of the seedlings and cultivate robust autotrophic seedlings.

[0006] (5) After completing the above full cultivation cycle, you can obtain strong seedlings of *Deer Antler Grass* with complete rooting, which can be directly transplanted.

[0007] Furthermore, during the entire culture cycle of dark culture in step (3) and staged light culture in step (4), the sugar-free culture box adopts an intermittent circulating gas supply mode. CO2 gas is introduced into the filter air filling hole in the sugar-free culture box every 30 minutes. The duration of each air supply is 20-30 seconds, and the CO2 concentration is 680-700 ppm. The indoor CO2 concentration of sugar-free culture is preferably maintained at 680-700 ppm, replacing the traditional culture medium sucrose as the carbon source for the plant.

[0008] Furthermore, the sugar-free incubator measures 30cm*20cm*15cm. The leaves are placed flat on the substrate with a 1cm gap between them, with the front side facing up. The relative humidity inside the box is maintained at over 95%, and the temperature is kept constant at 27℃.

[0009] Furthermore, in step (3), the leaves are pretreated before inoculation by first dipping the cleaned leaves of *Deer Antler Grass* into a mixture of 600-700 mg / L alginate and 100-200 mg / L NAA for 30 seconds; the mixture is made by mixing the two solutions of the above-prepared concentrations at a volume ratio of 1:1.

[0010] Furthermore, in step (3), the end is dipped in a mixture of 600 mg / L phycocyanin and 200 mg / L NAA for 30 seconds.

[0011] The beneficial effects of this application are: This application proposes for the first time a sugar-free tissue culture substrate specifically for the light environment of *Deer Antler Grass* leaves. Specifically, a sugar-free substrate consisting of a 5:1 volume ratio of Buerg peat moss and perlite is used as an alternative to conventional solid culture media. Through multi-stage seedling cultivation, using leaves as explants, a 10-day dark culture process induces scab formation. This is followed by two stages of light environment control: low-light differentiation to promote root growth, and strong-light seedling strengthening, all under a constant temperature of 27℃. This allows for rapid seedling formation of *Deer Antler Grass* without the need for hardening-off, significantly shortening the seedling cycle and greatly improving the efficiency of large-scale seedling supply. Comparative experiments show that compared to traditional *Deer Antler Grass* sowing and seedling cultivation methods, the seedling cycle can be shortened from 10 months to 3 months, with a faster seedling rate. Furthermore, compared to conventional tissue culture containing sucrose, the substrate formula in this application promotes rapid seedling growth, eliminating the need for hardening-off after seedling formation and quickly meeting the needs of large-scale *Deer Antler Grass* cultivation.

[0012] 2. The seedling cultivation method of this application significantly reduces seedling costs and greatly decreases pollution and disease incidence in the tissue culture environment. This is because the entire process uses a sugar-free culture system, and intermittent CO2 replaces sucrose as the carbon source, eliminating the need for the traditional tissue culture mode using sucrose as a solid culture medium. Furthermore, we have developed an effective disinfection method suitable for the sugar-free substrate of this application, namely a standardized substrate disinfection process involving soaking in 0.1% potassium permanganate for 4 hours. This disinfection cost is lower than conventional methods such as carbendazim and high-pressure sterilization, and it is more effective at killing fungi and insect eggs in the substrate. Combined with the leaf pretreatment method, it can effectively improve the rooting and subsequent root development of *Deer Antler Grass*.

[0013] 3. This application proposes for the first time an environmental control method specifically for sugar-free tissue culture of *Deer Antler Grass*, utilizing the combined effects of light and CO2 to significantly improve seedling quality and transplant survival rate. Simultaneously, the three-stage light regulation, combined with intermittent CO2 supply, can rapidly induce direct rooting in *Deer Antler Grass* leaf tissue culture and promote root development, ultimately cultivating robust autotrophic seedlings. Comparative analysis shows that the transplant survival rate is significantly higher than that of conventional sugar-containing tissue culture seedlings. This asexual propagation method of *Deer Antler Grass* leaves avoids various problems of phenotypic segregation in seedlings, resulting in uniform seedling traits, stable medicinal quality, and suitability for large-scale standardized cultivation.

[0014] 4. Through extensive research, this invention has developed a specialized seedling substrate for *Deer Antler Grass*, using a mixture of 0-10mm Pinscher peat moss and perlite in a 5:1 volume ratio. Combined with a sterilization method, this formula balances the substrate's aeration and water retention during tissue culture, significantly improving seedling stability. It is particularly suitable for the delicate root systems of *Deer Antler Grass*, preventing waterlogging and root rot. Our comparative analysis shows that a mixture of 0-10mm Pinscher peat moss and a small amount of perlite allows for better contact between the *Deer Antler Grass* leaves and the substrate, promoting root development. Attached Figure Description

[0015] Figure 1 This application relates to a sugar-free tissue culture experiment on the light environment of *Deer Antler Grass* leaves. Figure 2 This application relates to a sugar-free light environment incubator for *Deer Antler Grass* leaves. Figure 3 This is a diagram illustrating the light environment tissue culture process of *Deer Antler Grass* leaves in this application; Figure 4 This is a diagram of sugar-free rooting of *Deer Antler Grass* leaves in this application; Figure 5 This is a diagram of the transplantation of *Deer Antler Grass* as described in this application. Detailed Implementation

[0016] The following will refer to the appendices in the embodiments of the present invention. Figure 1-5 The technical solutions in the embodiments of the present invention are clearly and completely described herein. Obviously, the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention. Example 1

[0017] A method for sugar-free tissue culture of *Deer Antler Grass* leaves under light conditions includes the following steps: Prepare the sugar-free base: Mix 0-10mm Pinsbury peat moss and perlite at a volume ratio of 5:1 until homogeneous; Disinfection pretreatment: Prepare a 0.1% potassium permanganate solution, and completely immerse the sugar-free matrix from step (1) in the potassium permanganate solution for 4 hours; (3) Leaf explant inoculation and dark culture: Select disease-free, mature and intact leaves of *Deer Antler Grass*; Before inoculation, pre-treat the leaves by dipping the ends of the washed leaves in a mixture of 600 mg / L alginate and 200 mg / L NAA for 30 seconds; the mixture is a 1:1 volume ratio of the two solutions prepared above; then inoculate the leaves into the sterilized sugar-free substrate of step (2); place them in a sugar-free incubator for continuous dark culture for 10 days; (4) Staged light cultivation: The first stage of low-light cultivation: After the dark cultivation is completed, the light is turned on and the light intensity is controlled at 1000 lx. The light is provided for 12 hours a day for 2 consecutive months. The second stage is the cultivation of strong seedlings under strong light: After the seedlings reach a height of 3cm, the light intensity is increased to 3000lx, with 12 hours of light per day, and this is continued for 20 days to strengthen the photosynthetic tissue of the seedlings and cultivate robust autotrophic seedlings.

[0018] (5) After completing the above full cultivation cycle, you can obtain strong seedlings of *Deer Antler Grass* with complete rooting, which can be directly transplanted.

[0019] In the entire culture cycle, including the dark culture in step (3) and the phased light culture in step (4), an intermittent circulating gas supply mode is used in the sugar-free culture box. CO2 gas is introduced into the filter air vents of the sugar-free culture box every 30 minutes, with each air supply lasting 20-30 seconds. The gas is indoor air, and the indoor CO2 concentration is maintained at 680-700 ppm, replacing the traditional sucrose culture medium as the carbon source for the plants. The size of the culture box is 30cm*20cm*15cm. The leaves are placed flat on the substrate with a 1cm gap, with the upper surface facing up. The relative humidity inside the box is maintained at approximately 95%, and the temperature is kept constant at 27℃. Example 2

[0020] A method for sugar-free tissue culture of *Deer Antler Grass* leaves under light conditions includes the following steps: Prepare the sugar-free base: Mix 0-10mm Pinsbury peat moss and perlite at a volume ratio of 5:1 until homogeneous; Disinfection pretreatment: Prepare a 0.1% potassium permanganate solution, and completely immerse the sugar-free matrix from step (1) in the potassium permanganate solution for 4 hours; (3) Leaf explant inoculation and dark culture: Select disease-free, mature and complete leaves of *Deer Antler Grass*; Before inoculation, pre-treat the leaves by dipping the ends of the washed leaves in a mixture of 600 mg / L alginate and 200 mg / L NAA for 30 seconds; the mixture is prepared by mixing the two solutions of the above concentrations at a volume ratio of 1:1; then inoculate into the sugar-free substrate sterilized in step (2); place in a sugar-free incubator for continuous dark culture for 10 days. The sugar-free incubator has a top cover with filter and ventilation holes, and the holes are equipped with a sterile-grade breathable membrane, preferably a modified polypropylene membrane with a sterile-grade pore size of 0.22 μm.

[0021] (4) Staged light cultivation: The first stage of low-light cultivation: After the dark cultivation is completed, the light is turned on and the light intensity is controlled at 1000 lx. The light is provided for 12 hours a day for 2 consecutive months. The second stage is the cultivation of strong seedlings under strong light: After the seedlings reach a height of 3cm, the light intensity is increased to 3000lx, with 12 hours of light per day, and this is continued for 20 days to strengthen the photosynthetic tissue of the seedlings and cultivate robust autotrophic seedlings.

[0022] (5) After completing the above full cultivation cycle, you can obtain strong seedlings of *Deer Antler Grass* with complete rooting, which can be directly transplanted.

[0023] During the entire culture cycle, including the dark culture in step (3) and the phased light culture in step (4), the CO2 concentration in the sugar-free culture chamber was maintained at 800-850 ppm, replacing sucrose in the traditional culture medium as the carbon source for the plants. The incubator was 30cm*20cm*15cm in size, with the leaves placed flat on the substrate at 1cm intervals, facing upwards. The relative humidity inside the chamber was maintained at approximately 95%, and the temperature was kept constant at 27℃. Example 3

[0024] A method for sugar-free tissue culture of *Deer Antler Grass* leaves under light conditions includes the following steps: Prepare the sugar-free base: Mix 0-10mm Pinsworth peat moss and perlite in a volume ratio of 3:1 until homogeneous; Disinfection pretreatment: Prepare a 0.1% potassium permanganate solution, and completely immerse the sugar-free matrix from step (1) in the potassium permanganate solution for 4 hours; (3) Leaf explant inoculation and dark culture: Select disease-free, mature and complete leaves of *Deer Antler Grass*; Before inoculation, pre-treat the leaves by first dipping the ends of the washed *Deer Antler Grass* leaves in a mixture of 600 mg / L alginate and 200 mg / L NAA for 30 seconds; the mixture is prepared by mixing the two solutions of the above concentrations at a volume ratio of 1:1; then inoculate into the sterilized sugar-free substrate of step (2); place in a sugar-free incubator for continuous dark culture for 10 days. The sugar-free incubator is a transparent box with a top cover with filter ventilation holes and filter filling holes. Both the filter ventilation holes and the filter filling holes are equipped with sterile-grade breathable membranes, preferably modified polypropylene membranes with a sterile-grade pore size of 0.22 μm.

[0025] (4) Staged light cultivation: The first stage of low-light cultivation: After the dark cultivation is completed, the light is turned on and the light intensity is controlled at 1000 lx. The light is provided for 12 hours a day for 2 consecutive months. The second stage is the cultivation of strong seedlings under strong light: After the seedlings reach a height of 3cm, the light intensity is increased to 3000lx, with 12 hours of light per day, and this is continued for 20 days to strengthen the photosynthetic tissue of the seedlings and cultivate robust autotrophic seedlings.

[0026] (5) After completing the above full cultivation cycle, you can obtain strong seedlings of *Deer Antler Grass* with complete rooting, which can be directly transplanted.

[0027] In the entire culture cycle, including the dark culture in step (3) and the phased light culture in step (4), an intermittent circulating gas supply mode is used in the sugar-free culture box. CO2 gas is introduced into the filter air vents of the sugar-free culture box every 30 minutes, with each air supply lasting 20-30 seconds. The gas is indoor air, and the indoor CO2 concentration is maintained at 680-700 ppm, replacing the traditional sucrose culture medium as the carbon source for the plants. The size of the culture box is 30cm*20cm*15cm. The leaves are placed flat on the substrate with a 1cm gap, with the upper surface facing up. The relative humidity inside the box is maintained at approximately 95%, and the temperature is kept constant at 27℃.

[0028] Experiment 1: Optimization of parameters for sugar-free tissue culture seedlings from the leaves of *Deer Antler Grass* Experimental Methods: This experiment was conducted in the laboratory of Jiangxi Environmental Engineering Vocational College. Figure 1-5 As shown, where Figure 2The sugar-free incubator of this application has a filter and air filling hole in the middle of the top cover and filter and air vents around the perimeter. The tissue culture method of Example 1 was used. This experiment adopted a progressive parameter optimization scheme, that is, the optimal environmental parameters obtained in the previous stage were used as the basic culture conditions for the next stage. The dark culture was set with different treatment levels of 0 (full light), 7, 10, 14 and 20 days. The dark culture mainly counted the basal callus formation rate and root primordia induction rate. After the dark culture, the plants were uniformly transferred to a low light environment of 1000 lx and 12 h / d for 20 days. The rooted leaves of *Deer Antler Grass* with consistent growth obtained from the 10th day of the previous stage were selected and inoculated into a pretreated 0-10 mm mixture of Pints ​​peat and perlite in a 5:1 ratio. The light intensity and photoperiod gradient experiment was carried out and the plants were cultured continuously for 60 days. The rooting status and other related indicators were counted. Seedlings with a height of approximately 3 cm and uniform growth obtained in the previous stage were selected and kept under 12 h / d light. Four groups of seedling light intensity gradient designs were established, and the seedlings were cultured continuously for 20 days. Rooting and seedling vigor indicators were statistically analyzed. After cultivation, the seedlings were transplanted to a greenhouse substrate seedbed, and the 30-day transplant survival rate was measured. Experimental data were analyzed using SPSS 25.0 software for one-way ANOVA and Duncan's method for multiple comparisons. In addition, another independent experiment was set up with three control groups: Control Example 1 was conventional sugar-containing tissue culture, using 1 / 2 MS medium with 30 g / L sucrose added, with other conditions the same as Example 1, but without CO2 supply. After seedling establishment, the seedlings underwent 15 days of closed-flask culture followed by 10 days of hardening off before transplanting; Control Example 2 was constant light intensity sugar-free tissue culture, using a constant light intensity of 2000 lx without darkness, with substrate, CO2 concentration, and other conditions the same as Example 1; Control Example 3 had the temperature replaced by a cultivation temperature of 25℃, with other cultivation conditions the same as Example 1.

[0029] Experimental results: (1) As shown in Table 1, dark culture is a necessary condition for the dedifferentiation and scab formation at the base of *Desmodium styracifolium* leaves and the initiation of root primordia differentiation. In the 0d group without dark culture, the leaves were directly exposed to light, making it difficult for effective callus to form at the base, and the root primordia induction rate was less than 11%. As the dark culture time increased, the callus rate and root primordia induction first increased and then decreased, with the highest induction rate at 10d, uniform scab formation at the base, and the largest number of root primordia. After continuing dark culture for 14d, the leaves were in a heterotrophic state for a long time, and the tissue activity decreased, and the root primordia induction rate decreased significantly. Therefore, 10d is the optimal dark culture period for *Desmodium styracifolium*.

[0030] Table 1. Effects of dark incubation duration on callus and root primordia induction in *Pterocarya stenoptera* leaves.

[0031] Note: Different lowercase letters in the same column indicate significant differences (P<0.05). (2) As shown in Table 2-3, the low-light stage is the main stage for the development of root primordia into complete root systems and the sprouting and elongation of adventitious buds in *Deer Antler Grass*. Studies have found that light intensity and duration are the main factors affecting the rooting and development of *Deer Antler Grass* leaves. At a light intensity of 500 lx, insufficient light leads to low accumulation of photosynthetic products, weak root development, and thin, elongated seedlings. When the light intensity exceeds 1500 lx, newly emerging leaves are prone to photo-oxidative damage, resulting in leaf scorching and yellowing, and a decrease in both rooting and bud sprouting rates. An 8-hour photoperiod results in insufficient photosynthetic duration, low dry matter accumulation, and inhibition of both rooting and bud sprouting. A 16-hour photoperiod exceeds the effective photosynthetic duration of *Deer Antler Grass*, instead causing light stress and inhibiting seedling growth. Therefore, a low-light combination of 1000 lx and 12 h / d is selected for this stage, resulting in higher rooting and adventitious bud sprouting rates for *Deer Antler Grass*.

[0032] Table 2. Effects of different low light intensities on rooting and shoot development.

[0033] Table 3. Effects of different photoperiods on rooting and shoot development.

[0034] (4) As shown in Table 4, the seedling growth indicators of *Deer Antler Grass* in the treatment group with a light intensity of 3000 lx were significantly higher than those in other treatment groups, and its transplant survival rate reached the highest of 91.7%. When the light intensity was below 3000 lx, the seedlings had less photosynthetic accumulation and insufficient root development, failing to meet the standard for robust seedlings; when the light intensity exceeded 4000 lx, the photosynthetic efficiency of the leaves also decreased, resulting in a lower transplant survival rate. This indicates that seedlings under intermittent CO2 circulation gas supply, combined with a certain light intensity, can fully promote seedling development, and the second stage of cultivation can complete the process of robust seedlings in 20 days.

[0035] Table 4. Effects of different light intensities on rooting and transplant survival rates of vigorous seedlings.

[0036] As shown in Table 5, the sugar-free tissue culture method of Example 1 of this application resulted in the shortest seedling growth cycle for *Deer Antler Grass*, significantly shorter than that of conventional sugar-containing tissue culture media. However, the contamination rate was significantly lower than the sugar-containing control group, and the seedling quality was also higher. Compared to the constant light intensity of Control Example 2 and the constant temperature of 25℃ in Control Example 3, the environmental temperature of 27℃ and the phased gradient light in this application's examples were more suitable for *Deer Antler Grass* tissue culture seedlings. The seedlings had more developed root systems, a shorter seedling growth cycle, and a higher transplant survival rate. This was an unexpected discovery during our years of research on *Deer Antler Grass* tissue culture technology. A comparison of Examples 1-3 revealed that increasing the intermittent CO2 supply concentration and decreasing the proportion of Pints ​​peat perlite both led to an increase in the contamination rate and a decrease in the rooting rate of *Deer Antler Grass*.

[0037] Table 5 Comparison of growth indicators of *Deer Antler Grass* under different seedling raising techniques

[0038] Experiment 2: Optimization Experiment of Sugar-Free Tissue Culture Substrate Sterilization and Leaf Pretreatment of Deer Antler Grass Experimental Methods: This experiment was a preliminary preparation experiment for tissue culture materials. We compared the effects of different disinfection methods on the substrate disinfection of sugar-free tissue culture of *Deer Antler Grass*, and measured the contamination rate and disease rate. The experiment set up 3 experimental cases and 2 control groups, which was a gradient comparison experiment for the potassium permanganate disinfection method of this application. Experimental case 1 was to soak the substrate in 0.2% potassium permanganate solution for 4 hours, experimental case 2 was to soak the substrate in 0.1% potassium permanganate solution for 2 hours, experimental case 3 was to soak the substrate in 0.1% potassium permanganate solution for 5 hours, control case 4 was to replace the disinfectant in example 1 with 0.2% carbendazim wettable powder solution, and control case 5 was to use high-pressure steam sterilization, specifically high-pressure steam sterilization at 121℃ and 0.1MPa for 20 minutes, and then use it directly after cooling. Further optimization was performed on different leaf pretreatments, and rooting rate, root number, and root length were measured. Control Example 6 involved pretreatment of the explants by immersion in 75% ethanol for 30 seconds; Control Example 7 involved dipping the explant tip in a 600 mg / L phycocyanin solution for 30 seconds; Control Example 8 involved dipping the explant tip in a 200 mg / L NAA mixture for 30 seconds; Control Example 9 involved dipping the explant tip in a mixture of 500 mg / L phycocyanin and 50 mg / L NAA for 30 seconds; and Control Example 10 involved dipping the explant tip in a mixture of 600 mg / L phycocyanin and 200 mg / L NAA for 30 seconds. Except for the substrate disinfection and leaf explant pretreatment methods, all other culture conditions and environmental parameters remained the same. All treatments were repeated three times per group, with each replicate using 500 g of substrate and 30 leaves of *Pterocarya stenoptera*.

[0039] Table 6. Effects of different substrate disinfection treatments on sugar-free tissue culture of *Deer Antler Grass*

[0040] Experimental results: As shown in Table 6, we compared the potassium permanganate sterilization method of Example 1 of this application with several other sterilization methods and found that, under the same conditions, the contamination rate of high-pressure steam sterilization and potassium permanganate disinfection was low, while the contamination rate of carbendazim soaking was high. This indicates that the method is not thorough in killing endophytic fungi and spores in the substrate and cannot meet the sugar-free tissue culture requirements of deer antler grass.

[0041] Under the same 4-hour soaking condition, increasing the potassium permanganate concentration from 0.1% to 0.2% only slightly reduced the total contamination rate from 3.3% to 2.8%. The sterilization effect was optimal at 4 hours of soaking; further extending the soaking time did not provide any additional improvement, indicating that the 0.1% potassium permanganate concentration sterilization method with 4-hour soaking achieves superior sterilization results. Furthermore, the fungal diseases in seedlings are directly related to the initial bacterial load in the substrate; the disease rate reached 6.8% in the 2-hour soaking group.

[0042] Table 7 Effects of different leaf treatments on sugar-free tissue culture of *Deer Antler Grass*

[0043] Table 7 shows that this study found that ethanol disinfection of *Deer Antler Grass* leaves severely affected rooting. Specifically, the rooting rate of Control Example 6, which used ethanol for surface disinfection, was only 62.5%, with an average root count far lower than Example 1 and a root length of only 1.7 cm. This may be because explant sterilization led to weakened seedling growth and insufficient accumulation of photosynthetic products, failing to support root development. However, we found that the synergistic effect of rooting pretreatment, specifically the combination of phycocyanin and NAA, was the best. Its rooting rate, average root count, and average root length were significantly better than treatments using either of these two reagents alone (Control Examples 7 and 8). NAA, as an auxin, primarily promotes the development of the taproot and lateral roots of *Deer Antler Grass*; phycocyanin enhances root vitality and promotes root elongation. By comparing the combination of IAA and phycocyanin, we observed that although the rooting rate did not decrease significantly, the treatment group inhibited the elongation of *Deer Antler Grass* seedling roots, with an average root length significantly lower than Example 1.

[0044] Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.

Claims

1. A sugar-free leaf blade light environment tissue culture seedling raising method of Sabia japonica, characterized in that, Specifically, the steps include the following: (1) Prepare sugar-free matrix: Mix peat and perlite in a volume ratio of (3-8):1 until uniform; (2) Disinfection pretreatment: Prepare a 0.1% potassium permanganate solution and immerse the sugar-free matrix from step (1) completely in the potassium permanganate solution; (3) Leaf explant inoculation and dark culture: Select disease-free, mature, and intact leaves of *Deer Antler Grass* and inoculate them into the sterilized sugar-free substrate of step (2); place them in a sugar-free incubator for continuous dark culture for 10 days. (4) Staged light cultivation: The first stage is low light cultivation: After the dark cultivation is completed, the light is turned on and the light intensity is controlled at 800-1200 lx, with 12 hours of light per day; The second stage of strong light seedling cultivation: After the seedlings reach a height of 3cm, the light intensity is increased to 2000-4000lx, with 12 hours of light per day; (5) After completing the above full cultivation cycle, you can obtain strong seedlings of *Deer Antler Grass* with complete rooting, which can be directly transplanted.

2. The sugar-free leaf blade photoperiodic tissue culture seedling raising method of the genus Epimedium according to claim 1, characterized in that, The specific step (1) involves 0-10mm Pins peat, which is mixed with perlite at a volume ratio of 5:

1.

3. The sugar-free leaf blade photoperiodic tissue culture seedling raising method of Sagittaria trifolia according to claim 1, characterized in that, The matrix in step (2) is soaked in potassium permanganate solution for 4 hours.

4. The sugar-free leaf blade photoperiodic tissue culture seedling raising method of Sagittaria trifolia according to claim 1, characterized in that, The first stage of the weak light cultivation in step (4) lasts about 2 months, and the second stage of strong light seedling cultivation lasts 20 days.

5. The method for cultivating sugar-free tissue culture seedlings of *Deer Antler Grass* leaves according to claim 1, characterized in that, The sugar-free incubator is a transparent box with a top cover and filter air inlet. The filter air inlet is equipped with a sterile-grade breathable membrane, preferably a modified polypropylene membrane with a sterile-grade pore size.

6. The method for cultivating sugar-free tissue culture seedlings of *Deer Antler Grass* leaves according to claim 1, characterized in that, During the entire culture cycle of dark culture in step (3) and staged light culture in step (4), the sugar-free culture specifically adopts an intermittent circulating gas supply mode. Every 30 minutes, CO2 gas with a concentration of 680-700ppm is introduced into the filter air filling hole in the sugar-free culture box. The duration of each air supply is 20-30 seconds, and the cycle is repeated to replace the traditional culture medium sucrose as the carbon source for the plant.

7. The method for cultivating sugar-free tissue culture seedlings of *Deer Antler Grass* leaves according to claim 6, characterized in that, The relative humidity inside the box is maintained at approximately 95%, and the temperature in the culture room is kept constant at 27°C.

8. The method for cultivating sugar-free tissue culture seedlings of *Deer Antler Grass* leaves according to claim 1, characterized in that, In step (3), the leaves are pretreated before inoculation by first dipping the cleaned leaves of *Deer Antler Grass* in a mixture of 600-700 mg / L alginate and 100-200 mg / L NAA for 30 seconds. The mixture is prepared by mixing the two solutions of the above concentrations at a volume ratio of 1:

1.

9. The method for cultivating sugar-free tissue culture seedlings of *Deer Antler Grass* leaves according to claim 1, characterized in that, In step (3), the end is dipped in a mixture of 600 mg / L phycocyanin and 200 mg / L NAA for 30 seconds.

10. The method for cultivating sugar-free tissue culture seedlings of *Deer Antler Grass* leaves according to claim 1, characterized in that, The light intensity for the first stage of weak light cultivation in step (4) is 1000 lx; ​​the light intensity for the second stage of strong light seedling cultivation is 3000 lx.

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