Tissue culture and rapid propagation method for trichosanthes kirilowii maxim stem

Abstract

The invention relates to a Trichosanthes kirilowii Maxim stem tissue culture rapid propagation method, and belongs to the technical field of medicinal plant propagation. The method solves the problems of high explant pollution rate, low propagation efficiency, uneven seedling quality and dependence on expensive hormones or specific explants in the existing Trichosanthes kirilowii Maxim tissue culture. According to the technical scheme, the method is characterized in that tender stems of female trichosanthes kirilowii plants are used as explants, the explants are sterilized through ethyl alcohol and mercury bichloride, then the explants are inoculated into an MS culture medium containing 6-BA and NAA with the specific concentration to induce calluses, then the explants are transferred into a culture medium containing 6-BA with the high concentration and NAA with the low concentration to differentiate adventitious buds, and finally the explants are induced to root in a culture medium containing IBA to form complete plants. The method is mainly used for rapid and large-scale propagation of excellent trichosanthes kirilowii seedlings, especially female plants, and provides a stable seed source for production of traditional Chinese medicinal materials.

Description

Technical Field

[0001] The present invention belongs to the technical field of medicinal plant propagation. More specifically, the present invention relates to a method for rapid propagation of Trichosanthes kirilowii Maxim stem segment tissue culture. Background Art

[0002] Trichosanthes kirilowii Maxim Trichosanthes kirilowii Maxim is a perennial climbing herb of the Cucurbitaceae family. Its roots, fruits, pericarp, and seeds are all traditional Chinese medicinal materials, with the effects of clearing heat and promoting fluid production, resolving phlegm and relieving cough, and moistening the intestines and relieving constipation. It has wide clinical applications and significant medicinal value. Due to over-excavation and ecological environment destruction, the wild resources of Trichosanthes kirilowii Maxim have decreased sharply, and artificial cultivation has become the main way to ensure the supply of medicinal materials.

[0003] At present, the traditional propagation of Trichosanthes kirilowii Maxim mainly relies on seed propagation and root division propagation. In seed propagation, since Trichosanthes kirilowii Maxim is dioecious, gender identification cannot be carried out during the seedling stage, and the proportion of female plants is often low (female: male is about 3:7) after planting, resulting in unstable yields of medicinal fruits. In root division propagation, it requires the consumption of medicinal roots, with high costs, low propagation coefficients, and is prone to virus accumulation and varietal degeneration, making it difficult to meet the needs of large-scale production of high-quality seedlings.

[0004] Plant tissue culture technology has become an important means to solve the shortage of medicinal plant seedlings due to its advantages such as short propagation cycle, being not restricted by seasons, and enabling stable propagation of excellent genotypes. In the prior art, there have been studies exploring the tissue culture of Trichosanthes kirilowii Maxim: One type of research uses axillary bud stem segments or terminal buds as explants, and induces axillary buds to directly germinate into seedlings by adding lingfensin (LFS) to the culture medium. However, LFS is expensive (about 280 yuan / mg), relies on imports, and has unknown chemical components, which is not conducive to the standardization and industrialization promotion of the technology. More importantly, this method highly depends on specific buds with high physiological activity, and the material collection is severely restricted by seasons and plant states, making it impossible to achieve annual and large-scale production.

[0005] Another type of research attempts to use ordinary stem segments as explants to broaden the source of explants. For example, a researcher reported "Research on the Tissue Culture of Trichosanthes kirilowii Maxim" in the Journal of West Anhui University in April 2005, which recorded that Trichosanthes kirilowii Maxim stem segments can induce callus on the culture medium of MS + NAA 1.0 mg / L + 6-BA 0.5 mg / L, and green seedlings can be differentiated after 35 days of subculture. However, this type of method has obvious deficiencies: Low efficiency and unreliable: The above-mentioned scheme has a long differentiation period (more than 35 days), and only stays at the qualitative description level, lacking key quantitative effects, making it difficult to obtain stability and repeatability.

[0006] The technical approach is crude: callus induction and differentiation are carried out in the same culture medium, and the hormone ratio (high NAA / low 6-BA) is more conducive to callus growth than bud differentiation, resulting in extremely low differentiation efficiency.

[0007] There are common technical bottlenecks: Regeneration systems using ordinary stem segments as explants have long faced unresolved technical problems such as difficulty in surface sterilization, high contamination rate, low callus induction rate, poor adventitious bud differentiation efficiency, uneven seedling quality, and poor stability.

[0008] Therefore, there is an urgent need in this field to establish a rapid propagation method for Trichosanthes kirilowii tissue culture that has a wide range of explant sources, a clear hormone system, low cost, stable operation, and high regeneration efficiency, in order to overcome the shortcomings of existing technologies and realize the large-scale and standardized production of high-quality Trichosanthes kirilowii seedlings, especially female seedlings. Summary of the Invention

[0009] One object of the present invention is to address at least the aforementioned deficiencies and to provide at least the advantages that will be described later.

[0010] One objective of this invention is to provide a method for rapid propagation of Trichosanthes kirilowii stem segment tissue culture. By systematically optimizing the selection of explants, sterilization procedures, hormone ratios, light conditions, and culture system, this method effectively solves the problems existing in the prior art, such as high contamination rate, low regeneration efficiency, inconsistent seedling quality, and dependence on expensive hormones or specific explants.

[0011] This invention provides a method for rapid propagation of Trichosanthes kirilowii stem segments through tissue culture, using tender stem segments of female Trichosanthes kirilowii plants as explants, and includes the following steps: (1) Sterilization of explants: The tender stem segments were sterilized with 75% ethanol solution for 60 seconds and then sterilized with 0.1% mercuric chloride solution for 8 minutes to obtain sterile explants; (2) Callus induction: The sterile explants obtained in step (1) were inoculated into callus induction medium and cultured for 14 days at 23~27℃, light intensity of 1500~2500 lx and light duration of 14 h / d to induce callus formation; the callus induction medium was MS medium supplemented with 1.5~2.0 mg / L of 6-BA, 0.1~0.4 mg / L of NAA, 30 g / L of sucrose and 6 g / L of agar, with a pH of 5.8; (3) Adventitious bud differentiation: The callus induced in step (2) was transferred to the adventitious bud differentiation medium and cultured for more than 30 days under the same temperature and light conditions to induce the differentiation of adventitious buds; the adventitious bud differentiation medium was MS medium supplemented with 1.0~4.0 mg / L 6-BA, 0.1~0.4 mg / L NAA, 30 g / L sucrose and 6 g / L agar, with a pH of 5.8; (4) Rooting culture: The adventitious buds obtained in step (3) are cut off and transferred into the rooting medium. They are cultured for 14 days under the same temperature and light conditions to induce rooting and form complete regenerated plants. The rooting medium is MS medium with IBA 0.1~0.4 mg / L, sucrose 30 g / L and agar 6 g / L added, and the pH is 5.8.

[0012] Preferably, a method for rapid propagation of Trichosanthes kirilowii stem segments through tissue culture includes the following steps: (1) Sterilization of explants: The tender stem segments were sterilized with 75% ethanol solution for 60 seconds and then sterilized with 0.1% mercuric chloride solution for 8 minutes to obtain sterile explants; (2) Callus induction: The sterile explants obtained in step (1) were inoculated into the callus induction medium and cultured for 14 days at 23-27℃, light intensity of 1500-2500 lx and light duration of 14 h / d to induce callus formation; the callus induction medium was MS medium supplemented with 2.0 mg / L 6-BA, 0.1 mg / L NAA, 30 g / L sucrose and 6 g / L agar, with a pH of 5.8. (3) Adventitious bud differentiation: The callus induced in step (2) was transferred to the adventitious bud differentiation medium and cultured for more than 30 days under the same temperature and light conditions to induce the differentiation of adventitious buds; the adventitious bud differentiation medium was MS medium with 4.0 mg / L 6-BA, 0.1 mg / L NAA, 30 g / L sucrose and 6 g / L agar added, and the pH was 5.8. (4) Rooting culture: The adventitious buds obtained in step (3) are transferred to the rooting medium and cultured for 14 days under the same temperature and light conditions to induce rooting and form complete regenerated plants; the rooting medium is MS medium with IBA 0.4 mg / L, sucrose 30 g / L and agar 6 g / L added, and the pH is 5.8; or the rooting medium is MS medium with NAA 0.1 mg / L, IBA 0.4 mg / L, sucrose 30 g / L and agar 6 g / L added, and the pH is 5.8.

[0013] Preferably, in steps (2) and (3), the light conditions for cultivation are as follows: using an LED light source with a red light to blue light ratio of 3:1 to 5:1 and a light intensity of 2000 lx.

[0014] Preferably, in step (2), the callus induction culture medium also contains 0.5 mg / L of gibberellin GA3.

[0015] Preferably, the differentiation of adventitious buds in step (3) is carried out in a biphasic culture medium; the preparation method of the biphasic culture medium is as follows: first, dispense and autoclave the lower solid culture medium, and after it cools and solidifies, aseptically add the upper liquid culture medium to the container in a sterile operating table; The lower solid culture medium was MS medium supplemented with 4.0 mg / L 6-BA, 0.1 mg / L NAA, 30 g / L sucrose and 7.5 g / L agar, with a pH of 5.8; The upper liquid culture medium was MS medium supplemented with 4.0 mg / L 6-BA, 0.1 mg / L NAA and 30 g / L sucrose, with a pH of 5.8; The ratio of the volume of the upper liquid culture medium to the volume of the lower solid culture medium is 1:3 to 1:4.

[0016] Preferably, 0.1 mg / L of gibberellin GA3 is added to the upper liquid culture medium.

[0017] The present invention has at least the following beneficial effects: This invention uses ordinary young stem segments as explants, significantly reduces the contamination rate through a two-step sterilization method using ethanol and mercuric chloride, induces callus tissue with a specific ratio of 6-BA and NAA, promotes adventitious bud differentiation with high concentration of 6-BA, and finally induces rooting with IBA to form a complete regeneration system. This achieves the technical effects of wide availability of explants, low hormone cost, stable operation, and high regeneration efficiency.

[0018] This invention also replaces ordinary white light with an LED light source with a red-blue light ratio of 3:1 to 5:1. By regulating the photomorphogenesis and endogenous hormone balance of plants through specific light quality, it promotes the synchronous and uniform growth of callus tissue and adventitious buds, thereby improving the uniformity and robustness of tissue culture seedlings.

[0019] This invention also accelerates callus formation and improves its texture by adding a low concentration of gibberellin GA3 to the callus induction medium. This is achieved by breaking explant dormancy, promoting cell elongation and division, and working synergistically with 6-BA and NAA, making the callus more porous and uniform, thus laying the foundation for subsequent efficient and synchronized differentiation of adventitious shoots.

[0020] This invention also employs a dual-phase culture system, where the lower layer provides support and anchoring for the solid culture medium, while the upper layer provides a 360-degree environment for the absorption of nutrients and hormones without any dead angles for the liquid culture medium. This avoids growth differences caused by uneven contact in the solid culture medium, significantly improves nutrient utilization efficiency and growth uniformity, and balances aeration and water supply to promote robust seedling growth.

[0021] The present invention further promotes full water absorption and expansion of cells by adding a low concentration of GA3 to the upper liquid culture medium, thereby enhancing the robustness and uniformity of adventitious shoots and improving the overall culture quality.

[0022] Other advantages, objectives and features of the present invention will become apparent in part from the following description, and in part from those skilled in the art through study and practice of the invention. Attached Figure Description

[0023] Figure 1 The red frame indicates the tender stem segment of the female Trichosanthes kirilowii plant explant used in this invention.

[0024] Figure 2 This invention relates to the adventitious buds induced from female Trichosanthes kirilowii explants on an adventitious bud differentiation medium containing 4.0 mg / L 6-BA and 0.1 mg / L NAA during the adventitious bud differentiation stage, and their growth status.

[0025] Figure 3 This invention presents comparative images of adventitious buds induced from female *Trichosanthes kirilowii* explants on different hormone ratios during the adventitious bud differentiation stage, and their growth status. In the images, A: 4.0 mg / L 6-BA + 0.2 mg / L NAA; B: 4.0 mg / L 6-BA + 0.3 mg / L NAA; C: 2.0 mg / L 6-BA + 0.1 mg / L NAA; D: 3.0 mg / L 6-BA + 0.1 mg / L NAA.

[0026] Figure 4 This invention presents a comparative diagram showing the effects of different hormone ratios on rooting culture media during the rooting stage, illustrating the influence of these hormones on rooting. E: 0.3 mg / L NAA; F: 0.1 mg / L NAA + 0.1 mg / L IBA; G: 0.2 mg / L NAA + 0.3 mg / L BA; H: 0.1 mg / L NAA + 0.2 mg / L IBA; I: 0.1 mg / L NAA + 0.3 mg / L IBA; J: 0.1 mg / LNAA + 0.4 mg / L IBA.

[0027] Figure 5 This diagram illustrates the effects of different rooting medium ratios on rooting culture during the rooting stage, as described in this invention. A: 0 mg / L IBA (control); B: 0.1 mg / L IBA; C: 0.2 mg / L IBA; D: 0.3 mg / L IBA; E: 0.4 mg / L IBA. Detailed Implementation

[0028] The present invention will be further described in detail below with reference to embodiments, so that those skilled in the art can implement it based on the description.

[0029] It should be noted that, unless otherwise specified, the experimental methods described in the following implementation plan are all conventional methods, and the reagents and materials described are all commercially available unless otherwise specified.

[0030] This invention discloses a method for rapid propagation of Trichosanthes kirilowii stem segments through tissue culture, comprising the following steps: (1) Selection and surface sterilization of explants: Healthy, disease-free female Trichosanthes kirilowii stem segments (ordinary tender stem segments) were selected as explants, rinsed with running tap water and cut into small segments. They were sterilized in a clean bench by shaking with 75% ethanol and 0.1% mercuric chloride solution, and then rinsed with sterile water to effectively remove surface microorganisms and obtain sterile explants with low contamination rate and high survival rate. Under optimal conditions, the contamination rate after sterilization can be controlled below 10%.

[0031] (2) Callus induction: The sterile explants obtained in step (1) were inoculated into a medium based on MS medium and cultured for about 14 days at 23-27℃, light intensity of 1500-2500 lx, and photoperiod of 14 h / d to induce the formation of loose, granular callus. The medium was supplemented with 1.5-2.0 mg / L of 6-BA and 0.1-0.4 mg / L of NAA, 30 g / L of sucrose, and 6 g / L of agar, and the pH was adjusted to 5.8. Preferably, when the concentration of 6-BA was 2.0 mg / L and the concentration of NAA was 0.1 mg / L, the callus induction rate could reach 90±5%.

[0032] (3) Adventitious bud differentiation: The callus tissue induced in step (2) was transferred to a differentiation medium and cultured under the same environmental conditions for more than 30 days to induce the differentiation of multiple adventitious buds. The differentiation medium was MS-based, with 1.0~4.0 mg / L of 6-BA and 0.1~0.4 mg / L of NAA added, and the pH was adjusted to 5.8; preferably, the concentration of 6-BA was 4.0 mg / L and the concentration of NAA was 0.1 mg / L, which resulted in a high differentiation frequency and robust bud growth.

[0033] (4) Rooting culture: The adventitious buds obtained in step (3) are cut off and transferred to a rooting medium. Under the same environmental conditions, they are cultured for about 14 days to induce the formation of roots at the base of the adventitious buds, forming complete regenerated plants. The rooting medium is MS-based, with the addition of IBA 0.1–0.4 mg / L and / or NAA 0.1 mg / L, and the pH is adjusted to 5.8. The preferred IBA concentration is 0.4 mg / L, and the rooting rate can reach 100%.

[0034] Among them, 6-BA is 6-benzylaminopurine, NAA is naphthaleneacetic acid, and IBA is indolebutyric acid.

[0035] This invention establishes a stable and efficient indirect organogenesis and regeneration system by systematically optimizing the culture conditions at each stage.

[0036] In order to improve the uniformity of the differentiation of adventitious buds, the light conditions for cultivation in steps (2) and (3) can be replaced with LED light source with a light quality ratio of red light to blue light of 3:1 to 5:1 and light intensity of 2000 lx.

[0037] Unless otherwise specified, the light quality ratio mentioned in this invention refers to the ratio of photosynthetic photon flux density (PPFD).

[0038] Example 1 A method for rapid propagation of Trichosanthes kirilowii stem segments through tissue culture includes the following steps: A. Explant selection and surface sterilization Healthy, disease-free female Trichosanthes kirilowii stem segments were selected as explants. These segments were first rinsed under running tap water for 20-30 minutes, and then cut into small segments of approximately 2 cm. Next, in a clean bench, they were disinfected by immersion in a 75% ethanol solution for 60 seconds, followed by rinsing once with sterile water. Then, they were disinfected by immersion in a 0.1% mercuric chloride solution for 8 minutes, with gentle agitation to enhance sterilization. After sterilization, they were thoroughly rinsed 5 times with sterile water, each time for at least 1 minute, to completely remove residual disinfectant and obtain sterile explants.

[0039] B. Callus induction The sterile stem segments obtained in step A were inoculated onto callus induction medium. The medium consisted of MS medium containing 2.0 mg / L 6-BA, 0.1 mg / L NAA, 30 g / L sucrose, and 6 g / L agar, with a pH of 5.8. The culture conditions were: temperature 23–27°C, light intensity 2000 lx (e.g., provided by a cool white LED), and a photoperiod of 14 h / d. After 14 days of culture, a large amount of loose, granular, pale yellow callus tissue was obtained, with an induction rate of 90 ± 5%.

[0040] C. Adventitious bud differentiation The callus induced in step B was transferred to adventitious shoot differentiation medium. The medium consisted of MS medium containing 4.0 mg / L 6-BA, 0.1 mg / L NAA, 30 g / L sucrose, and 6 g / L agar, with a pH of 5.8. The culture conditions were the same as in step B. After 30 days of culture, a large number of adventitious shoots differentiated from the callus surface, with a differentiation rate reaching 100%.

[0041] D. Rooting culture: The adventitious shoots induced in step C were cut off from their base and transferred to rooting medium. The medium consisted of MS medium containing 0.4 mg / L IBA, 30 g / L sucrose, and 6 g / L agar, with a pH of 5.8. The culture conditions were the same as in step B. After 14 days of culture, numerous robust roots and fibrous roots formed at the base of the adventitious shoots, achieving a rooting rate of 100%.

[0042] The main reagents used in the experiment are shown in Table 1: Table 1: Test Reagents The main instruments used in the experiment are shown in Table 2: Table 2: Test Equipment or Instruments Example 1: Explant Screening Comparison Experiment 1 To verify the effectiveness and superiority of the Trichosanthes kirilowii stem segments used in this invention as explants in the rapid propagation method of tissue culture, the following comparative experiment 1 was set up to examine the callus induction ability, contamination rate and tissue tolerance of different explant types under the same sterilization and culture conditions.

[0043] Healthy, disease-free female Trichosanthes kirilowii plants were selected as experimental materials, and their tender leaves and stem segments were taken as candidate explants (see [link to experimental materials]). Figure 1 The explant pretreatment steps are as follows: First, rinse under running tap water for 20-30 minutes, and cut the stem segments into small segments of about 2 cm; then, in a clean bench, disinfect by immersing in a 75% ethanol solution for 60 seconds, and then rinse with sterile water 1-2 times; next, disinfect by immersing in a 0.1% mercuric chloride solution for 8 minutes, and gently agitate to improve the sterilization effect; after sterilization, rinse thoroughly with sterile water 3-5 times, each time for no less than 1 minute, to completely remove residual disinfectant and obtain sterile explants.

[0044] After sterilization, the leaf edges were trimmed, and the leaves were cut into approximately 1cm × 1cm pieces. For the stem segments, damaged tissue at both ends of the cut was removed. The treated leaf pieces and stem segments were inoculated into MS medium supplemented with 2.0 mg / L 6-BA, with 30 bottles in each group. The cells were cultured under the same conditions for 10 days, and the number of callus induced in each group was counted to determine the optimal explants.

[0045] The experimental results (see Table 3) show that when tender stem segments were used as explants, the callus induction rate reached 93.33%, the contamination rate was 6.67%, and obvious cell dedifferentiation was observed at the explant cut, forming pale yellow, loosely textured callus tissue with good growth. However, when tender leaf pieces were used as explants, no callus tissue was observed (induction rate was 0), and all inoculation bottles were contaminated with microorganisms (contamination rate 100.00%). The explants began to show browning, softening, and gradual decay on days 3 to 5 after inoculation.

[0046] Table 3: Effects of different explants on callus induction It is evident that, under the same sterilization procedures and culture conditions, tender stem segments outperformed tender leaf blocks in callus induction, contamination control, and explant survival. Therefore, tender stem segments are more suitable as explants for Trichosanthes kirilowii tissue culture.

[0047] Experiment 2: Screening and comparison of sterilization conditions in Example 1 To determine the optimal surface sterilization conditions for the *Trichosanthes kirilowii* stem segment explants in this invention, six different combinations of sterilization times were set up, and the specific operations are as follows: Stem segments from female *Trichosanthes kirilowii* plants were used as explants, cut into small segments approximately 2 cm long, rinsed under running water for 20-30 minutes, soaked in 75% ethanol (30 seconds, 60 seconds), rinsed once with sterile water, and then immersed in 0.1% mercuric chloride (6 minutes, 8 minutes, 10 minutes) with continuous shaking. The explants were then removed and rinsed five times with sterile water, each time for at least 1 minute, to thoroughly remove residual disinfectant and obtain sterile explants. The explants treated with different sterilization combinations were inoculated into MS medium without any added plant growth regulators, with 20 bottles inoculated per group. Under identical conditions, the inoculation status of each group was observed after 5 days, and the contamination rate was calculated using the following formula: Contamination rate = (Number of contaminated items / Number of inoculated items) × 100% The experimental results (see Table 4) show that different combinations of ethanol-mercuric chloride treatment times have a significant impact on the sterilization effect. When immersion in 75% ethanol for 60 seconds combined with treatment in 0.1% mercuric chloride for 8 minutes, the contamination rate is the lowest, at only 10%, and there is no obvious browning or softening of the explant incision, maintaining good turgor pressure and physiological activity. However, when the treatment time is too short (e.g., 75% ethanol for 30 seconds + 0.1% mercuric chloride for 6 minutes), the contamination rate is as high as 100%, and sterilization is incomplete. When the treatment time is too long (e.g., mercuric chloride for 10 minutes), the toxic damage to the explant is aggravated, with phenomena such as edge browning and tissue necrosis. Although the contamination rate is reduced, the survival rate is significantly decreased.

[0048] Table 4: Effects of different disinfection methods on contamination rate of the same type of explant It is evident that the treatment with 75% ethanol for 60 seconds combined with 0.1% mercuric chloride for 8 minutes resulted in the lowest contamination rate and good explant condition, representing the optimal sterilization conditions for this invention.

[0049] Comparative experiment 3 of inducing callus tissue in Example 1: Stem segments from healthy female *Trichosanthes kirilowii* plants were treated with the optimal sterilization conditions determined in Experiment 2 above. The sterilized stem segments, cut into 2cm pieces, were inoculated into MS medium supplemented with different concentrations of 6-BA (0, 0.5, 1.0, 1.5, 2.0 mg / L) and NAA (0, 0.1, 0.2, 0.3, 0.4 mg / L), resulting in 25 hormone combinations. Specific conditions are shown in Table 5. Each treatment was administered in 20 bottles, with 3 explants per bottle. Culture conditions were: temperature 23–27℃, light intensity 2000 lx, and photoperiod 14 h / d. Callus growth was observed and recorded every 3 days, and statistical analysis was performed after 14 days. Based on the time of callus appearance and callus growth, the optimal hormone ratio for callus induction medium was determined.

[0050] The experimental results (see Table 5) show that NAA alone was ineffective in inducing callus in *Trichosanthes kirilowii*, with a maximum induction rate of only 10.00%, and the induction effect was inhibited with increasing concentration. In contrast, 6-BA significantly promoted callus induction, and the induction rate increased with increasing concentration, reaching a maximum of 50.00%. When 6-BA and NAA were used in combination, the induction effect was significantly better than single hormone treatment. The combination of 2.0 mg / L 6-BA and 0.1 mg / L NAA in MS medium showed the best effect, with a callus induction rate as high as 90.00%.

[0051] Table 5: Effects of different hormones and their ratios on callus induction Note: The induction rate values ​​in the table are mean ± standard deviation; different lowercase letters in the same column indicate significant differences at the p=0.05 level; the same lowercase letter in the same column indicates no significant differences at the p=0.05 level.

[0052] It can be seen that MS medium + 2.0 mg / L 6-BA + 0.1 mg / L NAA is the most suitable medium for inducing callus from Trichosanthes kirilowii stem segments.

[0053] Comparative experiment 4 of callus induction of adventitious shoots in Example 1: Callus tissue successfully induced using the optimal callus induction medium (MS + 2.0 mg / L 6-BA + 0.1 mg / L NAA) determined in the comparative experiment 3 was inoculated into MS medium supplemented with different concentrations of 6-BA (1.0, 2.0, 3.0, 4.0 mg / L) and different concentrations of NAA (0, 0.1, 0.2, 0.3, 0.4 mg / L), resulting in 20 hormone combinations. Specific conditions are shown in Table 6. Each treatment involved 15 bottles of culture, with conditions identical to those in comparative experiment 3. Three 1cm × 1cm callus tissues were placed in each bottle. Callus differentiation was observed and recorded every 3 days, and statistical analysis was performed after 30 days. The number of adventitious shoots induced in each group of callus tissue was counted, and the differentiation rate was calculated to determine the optimal hormone ratio for callus differentiation medium.

[0054] Table 6: Effects of different hormones and concentration ratios on adventitious bud differentiation of Trichosanthes kirilowii Note: The induction rate values ​​in the table are mean ± standard deviation; different lowercase letters in the same column indicate significant differences at the p=0.05 level; the same lowercase letter in the same column indicates no significant differences at the p=0.05 level.

[0055] Callus clusters: Small (diameter ≤ 1cm); Medium (diameter 1cm < diameter < 2cm); Large (diameter ≥ 2cm) Buds: ≤2 scattered; 2 < 5 relatively dense; ≥5 dense Experimental results showed (see Table 6): the treatment combination B17 (MS + 4.0 mg / L 6-BA + 0.1 mg / L NAA) not only achieved the highest adventitious shoot differentiation rate (100.00%), but also induced large callus clusters with dense texture and numerous buds (see Table 6). Figure 2 and Figure 3 (Comparison). It can be seen that this combination is the optimal ratio of adventitious bud differentiation hormones in this invention.

[0056] Rooting induction comparative experiment 5 of Example 1: Adventitious shoots successfully differentiated from the optimal adventitious shoot differentiation medium (MS + 4.0 mg / L 6-BA + 0.1 mg / L NAA) determined in the above-mentioned comparative experiment 4 on callus induction of adventitious shoots were collected and inoculated into MS medium supplemented with different concentrations of NAA and IBA, resulting in a total of 25 hormone combinations. Specific conditions are shown in Table 7. Each treatment involved 15 bottles of culture, with the same conditions as in comparative experiment 3. Three adventitious shoots were placed in each bottle. The rooting status of the adventitious shoots was observed and recorded every 3 days. After 14 days, the results were statistically analyzed, including the number of roots, the number of lateral roots, the strength or weakness of the taproot, and the average taproot length, to determine the optimal hormone ratio for rooting.

[0057] Table 7: Effects of different hormones and their ratios on rooting Note: The induction rate values ​​in the table are mean ± standard deviation; different lowercase letters in the same column indicate significant differences at the p=0.05 level; the same lowercase letter in the same column indicates no significant differences at the p=0.05 level.

[0058] Main root: small (diameter ≤ 2mm); medium (diameter < 2mm < 5mm); robust (diameter ≥ 5mm) Rootlets: ≤5: few; 5 < < 8: relatively many; ≥8: many. The experimental results (Table 7) show that using an appropriate concentration of IBA alone yields the best results (see Table 7). Figure 5 Both rooting rate and root quality were significantly improved. Although NAA alone can also promote rooting rate and root quality to some extent, the synergistic effect of using NAA and IBA together is not significant. The amount of NAA added should not be too large, otherwise it will affect the rooting rate and root quality (see...). Figure 4 Among them, treatment combinations C4 (MS + 0.3 mg / L IBA) and C5 (MS + 0.4 mg / L IBA) both achieved a rooting rate of 100%. Further analysis of the root system revealed that treatment combination C5 had a thicker taproot and more fibrous roots, abundantly distributed at the bottom of the bottle, exhibiting the best overall performance (see...). Figure 5 E).

[0059] It is evident that MS + 0.4 mg / L IBA is the optimal rooting hormone ratio of this invention.

[0060] Example 2 A method for rapid propagation of Trichosanthes kirilowii stem segments by tissue culture is basically the same as in Example 1, except that in steps B and C, the light conditions for cultivation are replaced with an LED light source (such as a smart adjustable spectrum LED plant growth lamp) with a red light to blue light quality ratio of 3:1 and a light intensity of 2000 lx. The induction rate, differentiation rate, and rooting rate were approximately 96%, 100%, and 100%, respectively.

[0061] Example 3 A method for rapid propagation of Trichosanthes kirilowii stem segments by tissue culture is basically the same as in Example 1, except that in step B, 0.5 mg / L gibberellin GA3 is added to the callus induction medium. The induction rate, differentiation rate, and rooting rate were approximately 93%, 100%, and 100%, respectively.

[0062] Example 4 A method for rapid propagation of Trichosanthes kirilowii stem segments via tissue culture is basically the same as in Example 1, except that the differentiation of adventitious buds in step C is carried out in a biphasic culture medium; the specific operation of the biphasic culture medium is as follows: 1. Container preparation: Choose culture containers with good light transmittance and resistance to autoclaving, such as tissue culture glass bottles (e.g., 100mL volume) or disposable sterile plastic culture boxes. The containers should have a breathable sealing film or cap.

[0063] 2. Preparation and dispensing of the lower solid culture medium: Prepare the culture medium according to the formula (MS + 6-BA 4.0 mg / L + NAA 0.1 mg / L + sucrose 30 g / L + agar 7.5 g / L, pH 5.8).

[0064] Dispense the prepared culture medium solution into the final culture containers, with each container containing approximately 15-20 mL (for a 100 mL container, this should occupy about 1 / 3 to 1 / 2 of the container's height).

[0065] Cover with sealing film or lid (allowing steam to pass through) and autoclave (conditions are usually: 121°C, 20-30 min).

[0066] After sterilization, let it stand at room temperature outside the clean bench to allow it to cool naturally and solidify completely.

[0067] 3. Preparation and sterilization of the upper liquid culture medium: Prepare the liquid culture medium according to the formula (MS + 6-BA 4.0 mg / L + NAA 0.1 mg / L + sucrose 30 g / L, pH 5.8). Note: This formula does not include agar.

[0068] Dispense the prepared liquid culture medium into Erlenmeyer flasks and seal them with pressure-resistant caps or sealing film.

[0069] Perform high-pressure steam sterilization (under the same conditions as above).

[0070] After sterilization, transfer the conical flask containing the liquid culture medium to a clean bench and cool it to room temperature (about 30-40 degrees Celsius) for later use.

[0071] 4. Aseptically add the upper layer of liquid culture medium: In a clean bench, use aseptic techniques (such as flame flaming the bottle opening) to open the seal of the solidified lower layer of culture medium container.

[0072] Using a sterile pipette or sterile glass pipette / pipette, draw an appropriate amount of liquid from a sterilized and cooled conical flask containing liquid culture medium.

[0073] Slowly and gently add the liquid culture medium to the container that already contains the solid culture medium, allowing it to flow down the inner wall of the container, avoiding dispersing the surface of the solid culture medium.

[0074] Add approximately 5-7 mL (to make the total volume ratio of liquid to solid approximately 1:3 to 1:4).

[0075] 5. Vaccination: Once the upper liquid culture medium has been added and stabilized, the callus tissue block induced in step B can be gently placed on the surface of the lower solid culture medium using sterile forceps, so that it partially contacts the upper liquid culture medium.

[0076] Then, the culture was carried out under the established culture conditions.

[0077] The induction rate, differentiation rate, and rooting rate were approximately 92%, 100%, and 100%, respectively.

[0078] Example 5 A method for rapid propagation of Trichosanthes kirilowii stem segments by tissue culture is basically the same as that in Example 4, except that 0.1 mg / L gibberellin GA3 is added to the upper liquid culture medium in step C. The induction rate, differentiation rate, and rooting rate were approximately 91%, 100%, and 100%, respectively.

[0079] Example 6 A method for rapid propagation of *Trichosanthes kirilowii* stem segments by tissue culture is basically the same as in Example 5, except that in steps B and C, the light conditions are replaced with an LED light source (such as a smart adjustable spectrum LED plant growth lamp) with a red to blue light quality ratio of 3:1 and a light intensity of 2000 lx. In step B, 0.5 mg / L of gibberellin GA3 is also added to the callus induction medium. The induction rate, differentiation rate, and rooting rate were approximately 95%, 100%, and 100%, respectively.

[0080] Comparative Example 1 A method for rapid propagation of *Trichosanthes kirilowii* stem segments by tissue culture is basically the same as in Example 6, except that the formulation of the upper liquid culture medium in step C is different (MS + 6-BA 0.5 mg / L + NAA 1.0 mg / L + 0.1 mg / L gibberellin GA3 + sucrose 30 g / L, pH 5.8). The induction rate, differentiation rate, and rooting rate were approximately 95%, 39%, and 23%, respectively.

[0081] Comparative Example 2 A method for rapid propagation of Trichosanthes kirilowii stem segments by tissue culture is basically the same as in Example 1, except that the culture medium in steps B and C is MS + NAA 1.0 mg / L + 6-BA 0.5 mg / L + 30 g / L sucrose + 6 g / L agar. The induction rate, differentiation rate, and rooting rate were approximately 87%, 16%, and 7%, respectively.

[0082] Experiment 6 I. Experimental Procedure: Experimental materials: tender stem segments (2cm) from healthy, disease-free female Trichosanthes kirilowii plants from the same batch, with the same cut location and size.

[0083] Experimental grouping: The culture conditions of Examples 1-6 and Comparative Examples 1 and 2 were completely followed for grouping and culture, with 30 replicates in each group.

[0084] Comparative Example 2 (D2): MS + NAA 1.0 mg / L + 6-BA 0.5 mg / L.

[0085] Comparative Example 1 (D1): Unlike Example 6, the upper liquid culture medium was replaced with (MS + 6-BA 0.5 mg / L + NAA 1.0 mg / L + GA3 0.1 mg / L).

[0086] Example 1 (E1): The culture medium was prepared as follows: (callus induction medium MS + 6-BA 2.0 + NAA 0.1; adventitious shoot differentiation medium MS + 6-BA 4.0 + NAA 0.1; rooting medium MS + IBA 0.4), under normal white light.

[0087] Example 2 (E2): E1 + red and blue LEDs (3:1).

[0088] Example 3 (E3): E1+ callus induction medium with 0.5 mg / L GA3 added.

[0089] Example 4 (E4): Diphasic medium was used during the E1+ adventitious bud differentiation stage.

[0090] Example 5 (E5): GA3 0.1 mg / L was added to the upper layer of E4+ biphasic culture medium.

[0091] Example 6 (E6): E5 + red and blue LED (3:1) + GA3 0.5 mg / L added to callus induction medium.

[0092] II. Testing Methods and Evaluation Indicators: At the end of adventitious bud differentiation culture (30 days) and rooting culture (14 days), all surviving seedlings in each group were measured, and the mean, standard deviation (SD), and coefficient of variation (CV) were calculated. The smaller the standard deviation / coefficient of variation, the higher the uniformity and the better the stability.

[0093] 1. Adventitious bud stage (30 days of cultivation): Plant Height: The natural height (cm) of all adventitious buds in each group from the base to the tip.

[0094] Number of Effective Shoots: Count the number of seedlings with a height ≥2.0cm, fully expanded leaves, and vigorous growth that can be used for rooting in each group, and calculate the seedling survival rate (%).

[0095] Growth Status Score: Visually observed and scored based on the robustness of the seedlings, leaf color, and whether they have become vitrified (1-5 points, 5 points being the best).

[0096] 2. Rooting seedling stage (14 days of cultivation) Root Length: Measure the length (cm) of the longest root of all seedlings in each group.

[0097] Root System Score: The score is based on the number, thickness, and number of fibrous roots (1-5 points, with 5 points being the best).

[0098] Transplant Survival Rate: The survival rate (%) of rooted seedlings after transplanting them into a sterilized vermiculite-nutrient soil (1:1) substrate is calculated after 14 days. This is the standard for verifying the final stability and quality of the seedlings.

[0099] III. Test Results: Table 8: Growth uniformity data of adventitious buds (after 30 days of culture) Table 9: Quality and uniformity data of rooted seedlings (after 14 days of cultivation) As shown in Tables 8-9, Comparative Example 2 (D2) had the worst performance across all indicators, with coefficients of variation (CV) for seedling height and root length exceeding 50%, indicating extremely low uniformity. It also had the lowest seedling survival rate and transplant survival rate. The high NAA / low 6-BA hormone ratio favored callus growth rather than bud differentiation, leading to asynchronous differentiation and inconsistent adventitious bud quality. Weak seedlings and poor root development resulted in extremely poor post-transplant stability.

[0100] Although Comparative Example 1 (D1) used an advanced culture system (biphasic medium, LED, GA3), its uniformity (CV value) and seedling quality were far inferior to Example 1 (E1) due to the unreasonable hormone ratio (reverting to high NAA / low 6-BA). This shows that the hormone ratio is crucial, and other methods can only work synergistically on the basis of the hormone ratio.

[0101] Compared to D2, Example 1 (E1) shows a significant decrease in the CV values ​​of various indicators, and a substantial improvement in uniformity and stability, demonstrating the effectiveness of the core hormone ratio of this invention.

[0102] Example 2 (E2: E1 + LED) vs Example 3 (E3: E1 + GA3): Both had similar CV values, superior to E1, but with different improvement rates. E2 was slightly better in seedling height uniformity, while E3 was slightly better in seedling quantity. E2 (LED) more effectively regulated photomorphogenesis through a specific red-blue light (3:1) ratio, promoting the balance of endogenous hormones in plants, resulting in more synchronized and robust seedling growth, and reducing growth differences caused by light signal disturbances under ordinary white light. E3 (GA3) promoted uniform cell elongation and division through low-concentration GA3, breaking the dormancy state of some explants, making the callus tissue more homogeneous, and laying the foundation for subsequent synchronized differentiation.

[0103] Example 4 (E4: E1 + biphasic medium) showed a significant decrease in CV value and exhibited significantly better uniformity than E1, E2, and E3. This demonstrates that the biphasic medium is a key design feature for improving uniformity. The upper liquid medium ensures that each explant fragment can absorb nutrients and hormones evenly and without blind spots, completely avoiding the uneven growth problems caused by different contact positions on solid media, thus achieving extremely high synchronization of growth.

[0104] The CV value of Example 5 (E5: E4 + GA3) decreased further. The addition of low-concentration GA3, based on the homogeneous environment provided by the biphasic medium, further promoted uniform cell expansion and elongation, resulting in more consistent seedling growth.

[0105] Example 6 (E6) showed the lowest CV value among all groups, with high uniformity, and seedling survival rate and transplant survival rate both exceeding 99%, making it the best performing group.

[0106] Therefore, this invention effectively controls the contamination rate by optimizing the explant sterilization process, hormone ratio, light quality conditions, and biphasic culture system, significantly improving the callus induction rate, adventitious bud differentiation efficiency, and growth uniformity of regenerated seedlings. The resulting tissue culture seedlings have well-developed root systems, uniform growth, and high transplant survival rate, enabling the stable and large-scale production of female Trichosanthes kirilowii seedlings.

[0107] Although embodiments of the present invention have been disclosed above, they are not limited to the applications listed in the specification and embodiments. It can be applied to various fields suitable for the present invention. Further modifications can be readily implemented by those skilled in the art.

Claims

1. A method for rapid propagation of Trichosanthes kirilowii stem segments through tissue culture, characterized in that, Using tender stem segments from female Trichosanthes kirilowii plants as explants, the following steps were performed: (1) Sterilization of explants: The tender stem segments were sterilized with 75% ethanol solution for 60 seconds and then sterilized with 0.1% mercuric chloride solution for 8 minutes to obtain sterile explants; (2) Callus induction: The sterile explants obtained in step (1) were inoculated into callus induction medium and cultured for 14 days at 23~27℃, light intensity of 1500~2500 lx and light duration of 14 h / d to induce callus formation; the callus induction medium was MS medium supplemented with 1.5~2.0 mg / L of 6-BA, 0.1~0.4 mg / L of NAA, 30 g / L of sucrose and 6 g / L of agar, with a pH of 5.8; (3) Adventitious bud differentiation: The callus induced in step (2) was transferred to the adventitious bud differentiation medium and cultured for more than 30 days under the same temperature and light conditions to induce the differentiation of adventitious buds; the adventitious bud differentiation medium was MS medium supplemented with 1.0~4.0 mg / L of 6-BA, 0.1~0.4 mg / L of NAA, 30 g / L of sucrose and 6 g / L of agar, with a pH of 5.8; (4) Rooting culture: The adventitious buds obtained in step (3) are cut off and transferred into the rooting medium. They are cultured for 14 days under the same temperature and light conditions to induce rooting and form complete regenerated plants. The rooting medium is MS medium with IBA 0.1~0.4 mg / L, sucrose 30 g / L and agar 6 g / L added, and the pH is 5.

8.

2. The method according to claim 1, characterized in that, The rooting medium in step (4) also contains 0.1 mg / L NAA, resulting in an MS medium containing 0.1 mg / L NAA, 0.4 mg / L IBA, 30 g / L sucrose and 6 g / L agar, with a pH of 5.

8.

3. The method according to claim 1, characterized in that, The callus induction medium contains 2.0 mg / L of 6-BA and 0.1 mg / L of NAA; the adventitious shoot differentiation medium contains 4.0 mg / L of 6-BA and 0.1 mg / L of NAA; and the rooting medium contains 0.4 mg / L of IBA.

4. The method according to claim 1, characterized in that, In steps (2) and (3), the light conditions for cultivation are as follows: LED light source with a red light to blue light ratio of 3:1 to 5:1 and light intensity of 2000 lx is used.

5. The method according to any one of claims 1-4, characterized in that, In step (2), 0.5 mg / L of gibberellin GA3 is also added to the callus induction culture medium.

6. The method according to any one of claims 1-4, characterized in that, In step (3), the differentiation of adventitious buds is carried out in a biphasic culture medium. The preparation method of the biphasic culture medium is as follows: first, dispense and autoclave the lower solid culture medium, and after it cools and solidifies, aseptically add the upper liquid culture medium to the container under a sterile operating table. The lower solid culture medium was MS medium supplemented with 4.0 mg / L 6-BA, 0.1 mg / L NAA, 30 g / L sucrose and 7.5 g / L agar, with a pH of 5.8; The upper liquid culture medium was MS medium supplemented with 4.0 mg / L 6-BA, 0.1 mg / L NAA and 30 g / L sucrose, with a pH of 5.8; The ratio of the volume of the upper liquid culture medium to the volume of the lower solid culture medium is 1:3 to 1:

4.

7. The method according to claim 6, characterized in that, The upper liquid culture medium also contains 0.1 mg / L of gibberellin GA3.

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