Method for improving efficiency of artificial propagation of trichosanthes cucumerina var. grandiflora

Abstract

The present application relates to the technical field of plant induction regeneration, and particularly relates to a method for improving artificial propagation efficiency of Trichosanthes cucumerina var. villosa, which uses Trichosanthes cucumerina var. villosa stem segments as explants and optimizes the proliferation medium, fixes the excellent traits of the female parent to the greatest extent through direct organogenesis, avoids the variation possibly caused by indirect organogenesis, and has a proliferation cycle of 40 days and a propagation coefficient of more than 14.0; the method meets the needs of large-scale production of high-quality Trichosanthes cucumerina var. villosa seedlings, solves the bud tip necrosis phenomenon commonly existing in in vitro rapid propagation of cucurbitaceae plants, improves the quality of seedlings, and improves the economic benefits; the present application has important significance and value for artificial rapid propagation of Trichosanthes cucumerina var. villosa, and lays a technical foundation for protecting the wild resources and developing artificial planting.

Description

Technical Field

[0001] This invention belongs to the field of plant induced regeneration technology, and particularly relates to a method for improving the efficiency of artificial propagation of Trichosanthes kirilowii. Background Technology

[0002] Trichosanthes kirilowii ( Trichosanthes cucumerina Linn., belongs to the genus Trichosanthes of the family Cucurbitaceae. Trichosanthes It is an annual climbing vine; *Trichosanthes kirilowii* is rich in various chemical components, such as flavonoids, carotenoids, and phenolic acids, giving it high activity in pharmacology and therapeutics. et al. (2010). The medicinal parts of Trichosanthes kirilowii are diverse, including the fruit, leaves, roots, and seeds, and it has extremely high edible and medicinal value.

[0003] Due to large-scale habitat destruction and overexploitation, the wild resources of *Trichosanthes kirilowii* are facing depletion. Although artificial cultivation has been used to rescue *Trichosanthes kirilowii* germplasm resources, traditional seedling methods are unsuitable: firstly, the number of *Trichosanthes kirilowii* seeds is small and the germination rate is extremely low; secondly, in actual production, *Trichosanthes kirilowii* is often propagated asexually, such as by cutting and germinating root segments, which has a low propagation coefficient and requires a large number of mother plants, increasing production costs. It is precisely because of these limitations of conventional propagation methods that researchers are forced to seek a more efficient and stable method for propagating *Trichosanthes kirilowii*. Plant tissue culture technology is one of the important means to achieve germplasm innovation, germplasm resource propagation, and preservation, and it is also a major pathway and effective method for achieving industrialized seedling production of *Trichosanthes kirilowii*. Currently, research on in vitro rapid propagation of the *Trichosanthes* genus is mostly focused on optimizing the culture protocols for *Trichosanthes* plants, and only a small portion of the research is currently available. Trichosanthes dioica Roxb has reported on shoot tip necrosis (STN), a physiological disorder that causes yellowing and eventual death of the stem tips of in vitro seedlings. STN is a common and prevalent adverse phenomenon in plant tissue culture, severely impacting the growth, development, domestication, and subsequent technology promotion of in vitro seedlings. Its incidence and severity appear to be related to species differences, particularly affecting trees and woody shrubs. Common symptoms include senescence and death of terminal buds and young leaves, gradually extending towards the base. Secondary buds usually sprout from axillary buds in the non-necrotic parts, but with prolonged culture time, STN can also develop at the tips of these secondary buds, leading to a reduction in the number of high-quality buds, resulting in significant culture loss and increased production costs.

[0004] Due to the potential medicinal value and extremely low sexual reproduction efficiency of *Trichosanthes kirilowii*, many scholars have conducted research on its rapid in vitro propagation. However, significant problems remain in improving STN (strain-transplanted tissue nucleus), propagation coefficient, transplant survival rate, and high cost, keeping the overall application of tissue-cultured seedlings at a very low level. Therefore, there is a need to find a new, low-cost, short-time, high-quality, and high-survival-rate asexual reproduction method that is STN-free and can fix superior traits to expand the propagation volume of *Trichosanthes kirilowii* seedlings and enable the industrial production of high-quality seedlings to meet planting demands. Summary of the Invention

[0005] The purpose of this invention is to provide a method to improve the efficiency of artificial propagation of Trichosanthes kirilowii, which solves the problems of poor proliferation effect, STN in the bud propagation stage, resulting in a reduction in the number of high-quality buds, serious loss of culture and increase in production costs, difficulty in large-scale and standardized planting, and difficulty in promoting high-efficacy varieties on a large scale in the rapid in vitro propagation of Trichosanthes kirilowii.

[0006] To solve the above technical problems, the present invention adopts the following technical solution: The methods to improve the efficiency of artificial propagation of Trichosanthes kirilowii include the following steps: (1) Obtaining explants: Select healthy plants with good growth and no pests or diseases, and take stem segments with nodes; (2) Disinfect the stem segments with nodes from step (1) and cut them into appropriate sizes; In some preferred methods, after cleaning the explants, removing excess petioles and leaves, and trimming both ends of the cut, the explants are transferred to culture medium A for further culture. (3) The segmented stem that has been disinfected and sterilized in step (2) is inoculated into culture medium A for culture to obtain sterile test-tube seedlings. Culture medium A is based on MS medium, with 0.5-1.5 mg / L 6-benzylpurine, 30000 mg / L sucrose and 4700 mg / L agar powder added. In some preferred methods, when the explant is inserted into culture medium A, at least one node should be submerged in culture medium A. By controlling the light intensity, temperature and light duration during culture, a large number of clustered shoots can be obtained at the base of the culture. (4) Development and proliferation of basal shoot clusters: After cutting the sterile test-tube seedlings cultured in step (3) into appropriate sizes, they were transferred to culture medium B for further culture to obtain robust shoot clusters. Culture medium B includes: MS medium, 6-benzylpurine, kinetin, naphthaleneacetic acid, and Ca. 2+ ; (5) Take the STN-free clustered buds from step (4), and inoculate them individually in culture medium C to induce rooting. Culture medium C uses MS medium as the basic medium and adds 0.5-1.0 mg / L naphthaleneacetic acid. Specifically, when the height of the clustered buds in the optimized bud proliferation medium reaches 4 cm, they are divided into single buds and transferred to medium C. By controlling the light intensity, temperature, and light duration, rooting can be induced 100%. (6) Hardening off and transplanting: Take the rooted plants from step (5) and harden them off at room temperature. Take the seedlings out of culture medium C, clean off the residual culture medium, and disinfect them in carbendazim solution. (7) Transplant the disinfected rooted seedlings into the disinfected soil substrate and keep them warm and moist to obtain transplanted seedlings.

[0007] Furthermore, in step (1), the length of the segmented stem is 4-5 cm and the number of segments in the stem is >2.

[0008] Furthermore, the method for disinfecting the segmented stem in step (2) is as follows: rinse with running water for 4 hours, place on a clean bench and treat with 75% ethanol solution for 5-10 seconds, then sterilize with 0.1% mercuric chloride solution for 10-12 minutes, and finally rinse with sterile water 2-3 times, each time for no less than 2 minutes.

[0009] Furthermore, the environmental conditions in steps (3)-(6) are 25 ± 1℃, light intensity of 1500-1800 lx, and daily light exposure of 7-9 h.

[0010] Furthermore, the environmental conditions in step (7) are 20-28℃, humidity of 60%-80%, and light intensity of 2200-2600 lx.

[0011] Furthermore, the pH value of the culture medium in steps (3)-(6) is 5.6-5.8.

[0012] Furthermore, before transferring the sterile test-tube seedlings into culture medium B in step (4), they are cut into stem segments with a length of 4-5 cm and a number of nodes > 2.

[0013] Further, the culture medium B uses MS medium with adjusted total calcium and boron concentrations as the basal medium, supplemented with 2.0-4.0 mg / L 6-benzylpurine, or 2.5-3.5 mg / L 6-benzylpurine, or 2.5-3.0 mg / L 6-benzylpurine, or 2.8-3.5 mg / L 6-benzylpurine, or 3.0-3.2 mg / L 6-benzylpurine; and supplemented with 1.0-3.0 mg / L kinetin, or 1.5-2.5 mg / L kinetin, or 1.8-2.0 mg / L kinetin, or 1.8-3.0 mg / L kinetin, or 2.0-2.5 mg / L kinetin; and 0.1-1.0 mg / L naphthaleneacetic acid, or 0.1-0.8 mg / L naphthaleneacetic acid, or 0.2-0.6 mg / L naphthaleneacetic acid. Naphthaleneacetic acid, or 0.2-0.5 mg / L naphthaleneacetic acid, or 0.5-1.0 mg / L naphthaleneacetic acid; and Ca 2+ The total concentration was 240-480 mg·L. -1 or 300-480 mg·L -1 340-450 mg·L -1 340-400 mg·L -1 340-420 mg·L -1 360-380 mg·L -1 .

[0014] Using MS medium as the basic medium, a suitable culture medium formula for the proliferation of stem segments with nodes of Trichosanthes kirilowii was obtained by screening the types and concentrations of plant growth regulators and the inorganic salt strength of the culture medium. The proliferation coefficient of the culture under this formula can reach 14.68, and the incidence of STN is always less than 10% at a subculture interval of 40 days.

[0015] Furthermore, the mass concentration of the carbendazim solution in step (6) is 0.1-0.2%.

[0016] Furthermore, the soil matrix is ​​composed of coconut coir, humus, and perlite mixed in a ratio of 2:2:1 (v / v).

[0017] Compared with the prior art, the present invention has the following beneficial effects: (1) This invention uses plant tissue culture technology and uses the stem segments of Trichosanthes kirilowii with nodes as explants. Through direct organogenesis, the superior traits of the parent plant can be fixed to the greatest extent, avoiding the variations that may occur through indirect organogenesis. (2) This invention achieves the goal of efficient and rapid propagation. A propagation culture cycle is 40 days, and the propagation coefficient can reach more than 14.0. (3) By optimizing the propagation culture medium of the rapid propagation system of Trichosanthes kirilowii, the needs of large-scale high-quality Trichosanthes kirilowii seedling production are met, while the STN problem is solved at the same time. This is the most effective propagation method for its artificial rapid propagation, which improves the quality of seedlings and enhances economic benefits. (4) This invention is of great significance and value for the rapid artificial propagation of Trichosanthes kirilowii, and lays a technical foundation for protecting its wild resources and developing artificial cultivation. Attached Figure Description

[0018] Figure 1 Diagrams showing the various growth stages established for a sterile system; Among them, 1A shows the growth after 10 days of culture; 1B shows the growth after 15 days of culture; 1C shows the growth after 25 days of culture; and 1D shows the growth after 35 days of culture. Figure 2 The effects of different types of hormones on stem segment proliferation; Wherein, 2E is MS+6-BA; 2F is MS+KT; 2G is MS+NAA; 2H is MS+2,4-D; Figure 3 The proliferation coefficient of the segmented stem under the influence of different types and concentrations of hormones; Figure 4 The effects of inorganic salt concentration and subculture interval on STN and proliferation of shoot clusters in MS medium; Of which, 4A was 240.24 mg·L⁻¹. -1 Ca 2+ +1.08 mg·L -1 B 3+ ; 4B is 480.48 mg·L -1 Ca 2+ +1.08 mg·L -1 B 3+ ; 4C is 720.72 mg·L -1 Ca 2+ +1.08 mg·L -1 B 3+ ; 4D was 960.96 mg·L -1 Ca 2+ +1.08 mg·L -1 B 3+ ;4E is 2.16 mg·L -1 B 3+ +120.12 mg·L -1 Ca 2+ ; 4F is 4.32 mg·L -1 B 3+ +120.12 mg·L -1 Ca 2+4G is 6.48 mg·L -1 B 3+ +120.12 mg·L -1 Ca 2+ ; 4H is 8.64 mg·L -1 B 3+ +120.12 mg·L -1 Ca 2+ ; Figure 5 The effects of calcium and boron concentrations in MS medium on shoot STN and proliferation; Figure 6 Diagrams showing the various growth stages of propagation and seedling formation; Among them, 6A shows the growth after 10 days of culture; 6B shows the growth after 20 days of culture; 6C shows the growth after 30 days of culture; and 6D shows the growth after 40 days of culture. Figure 7 Diagrams showing the various growth stages of transplanted plants after domestication; Among them, 7E shows the growth of the plant after 15 days of rooting culture; 7F shows the growth of the plant 20 days after transplanting after the hardening-off work is completed; 7G shows the growth of the plant root system 20 days after transplanting; 7H shows the growth of the plant 40 days after transplanting; and 7I shows the growth of the plant 4 months after transplanting. Detailed Implementation

[0019] To enable those skilled in the art to better understand the technical solutions of the present invention, the present invention will be described in detail below with reference to specific embodiments. Experimental methods in the following embodiments that do not specify specific conditions are generally performed under conventional conditions or as recommended by the manufacturer. Unless otherwise specified, the test materials used in the following embodiments were purchased from conventional biochemical reagent stores. Unless otherwise stated, percentages and parts are by weight. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those familiar with the art. Furthermore, any methods and materials similar to or equivalent to those described herein can be applied to the present invention. The preferred embodiments and materials described herein are for illustrative purposes only.

[0020] The endpoints and any values ​​of the ranges disclosed herein are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of the various ranges, the endpoint values ​​of the various ranges and individual point values, and individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed herein.

[0021] The technical solution of the present invention will be further described in detail below with reference to specific embodiments and accompanying drawings. It should be understood that the following embodiments are only used to explain the present invention and are not intended to limit the present invention.

[0022] The reagents used in the following examples are as follows: The sterilization reagent mercuric chloride (HgCl2) used in the experiment was purchased from Guizhou Tongren Chemical Research Institute; plant growth regulators 2,4-dichlorophenoxyacetic acid (2,4-D), kinetin (KT), 1-naphthylacetic acid (NAA), indole-3-butyric acid (IBA), 6-benzylpurine [N-(Phenylmethyl)-9H-purin-6-amine (6-BA)], sucrose, agar, calcium chloride (CaCl2·2H2O), boric acid (H3BO3), and anhydrous ethanol were all analytical grade and purchased from Beijing Dingguo Biotechnology Co., Ltd.

[0023] Example 1: Material Sources and Establishment of a Sterile System Healthy, well-developed stem segments were selected as explants, each with at least two nodes. After rinsing the explants under running water for 4 hours, they were placed in a clean bench and rinsed with 75% ethanol solution (v / v) for 5 seconds, followed by immersion in 0.1% mercuric chloride solution (w / v) for 10 minutes for disinfection. The disinfected explants were then rinsed twice with autoclaved sterile water, each time for at least 2 minutes. After cleaning, excess petioles and leaves were removed, and the cut ends were trimmed before transferring the explants to culture medium A. Culture medium A consisted of MS medium as the basal medium, supplemented with 0.5-1.5 mg / L 6-benzylpurine, 30000 mg / L sucrose, and 4700 mg / L agar powder. When inserting the explants into the solid culture medium, at least one node should be submerged. The culture was maintained at 25 ± 1 ℃, with a light intensity of 1500-1800 lx, and 7-9 hours of light per day, with intervals of 5 days. d. Observe and record the growth of the explants (see...) Figure 1 ).

[0024] like Figure 1 As shown (scale bar = 2.0 cm), where, Figure 1 As can be seen in B, after 15 days of cultivation, lateral buds began to sprout, and obvious light green buds appeared; Figure 1 As can be seen in Figure C, a large number of axillary buds emerged after 25 days of cultivation, successfully inducing cluster buds; Figure 1 As can be seen in Figure D, after 35 days of culture, the axillary buds further elongated, the leaves unfolded, and the nodes and internodes became clearly distinguishable. At the same time, the part of the bud cluster in contact with the culture medium swelled.

[0025] Example 2: Investigating the effects of different types of hormones on stem segment proliferation 2.1 Effects of a single hormone on its proliferation: Plant hormones 6-BA, KT, NAA, and 2,4-D were added to MS basal medium, with concentrations of 0.5, 1.0, 2.0, and 3.0 mg·L⁻¹, respectively. -1 The disinfected stem segments with nodes were cultured in the above-mentioned culture medium at 25 ± 1℃, with a light intensity of 1500-1800 lx and a daily light exposure of 7-9 h. After 45 days, the growth was observed and recorded (see...). Figure 2 ).

[0026] like Figure 2 As shown (scale = 3.0 cm), Figure 2 As can be seen in E, when 6-BA is added, a large number of fine axillary buds regenerate at the stem nodes. After the axillary buds grow and develop further, the plant appears as a cluster. Figure 2 As can be seen from F, the addition of KT significantly elongated the interstitial space of the plants, and the size of the leaves and the thickness of the stems were both superior to those of other experimental groups; Figure 2 As can be seen in G, the plants rooted well after the addition of NAA. Figure 2 As can be seen from H, under the same culture period, 2,4-D, apart from producing a large amount of callus at the base of the stem segment, had a very weak effect on inducing multiple buds. From Figure 3 As can be seen, the proliferation coefficients of 6-BA and KT increase with increasing hormone concentration. When the concentrations of both 6-BA and KT are 3.0 mg·L⁻¹, the proliferation coefficients increase further. -1 The maximum proliferation coefficients within their respective experimental groups were obtained at specific times, reaching 9.02 and 4.67, respectively. However, NAA and 2,4-D at the same concentrations showed a different trend. With increasing NAA and 2,4-D concentrations, the proliferation coefficients first increased and then decreased. Specifically, when the 2,4-D concentration was 1.0 mg·L⁻¹, the proliferation coefficients were highest. -1 The proliferation coefficient was the highest at 1.23, which is only about 1 / 7 of the maximum proliferation coefficient of 6-BA.

[0027] 2.2 Bud induction and orthogonal experiment with proliferation hormones: Based on the results of the single-factor preliminary experiment, the tissue culture seedlings cultured for 45 days were cut into stem segments (about 4-5 cm) with at least 2 nodes and inoculated into a culture medium composed of different types and concentrations of plant hormones. An orthogonal design experiment was carried out, and the results are shown in Table 1.

[0028] Table 1

[0029] As can be seen from the table above, R A> R C > R B > R D The results indicate that 6-BA, KT, and NAA all have reliable effects on shoot induction, with the order of effect being 6-BA > NAA > KT. Analysis of variance shows that KT and NAA have no significant effect on shoot induction. P >0.05), 6-BA has a significant effect ( P <0.05).

[0030] Example 3: Investigating the effects of subculture cycle and inorganic salt concentration in culture medium on shoot proliferation process STN. The subculture interval was set to 40 days, and MS was used as the basal medium. The calcium content in the medium was adjusted. 2+ B 3+ Total concentration; the calcium source in the culture medium is provided by calcium chloride (CaCl2·2H2O), Ca 2+， The total concentrations were 120.12, 240.24, 480.48, 720.72, and 960.96 mg·L, respectively. -1 And B 3+ The total concentration was maintained at 1.08 mg·L⁻¹. -1 Secondly, the boron source is provided by boric acid (H3BO3), B 3+ The total concentrations were 1.08, 2.16, 4.32, 6.48, and 8.64 mg·L⁻¹, respectively. -1 At the same time, 120.12 mg·L -1 Ca 2+ To investigate the independent effects of the two elements mentioned above on STN development, plant growth was observed after 40 days of culture (see...). Figure 4 The incidence and proliferation coefficient of STN in plants under each treatment were statistically analyzed (see [reference]). Figure 5 ).

[0031] like Figure 5 As shown on the left Y-axis, add Ca 2+ and B 3+ Total concentration had a significant impact on alleviating STN. Among all treatments, Ca... 2+ The concentration was 480.48 mg·L⁻¹. -1 B 3+ The concentration was 1.08 mg·L⁻¹ -1 The combination effectively suppressed the occurrence of STN, with over 91% of buds showing no STN; however, Ca 2+ The concentration increased to 720.72 mg·L⁻¹ -1 At that time, the incidence of STN increased instead of decreasing, and the incidence of Ca continued to increase. 2+Concentration up to 960.96 mg·L -1 STN showed no decreasing trend, consistently remaining around 20%; at low Ca... 2+ At the horizontal level, B 3+ The concentration was 2.16 mg·L⁻¹ -1 The incidence of STN can be reduced from the control group (120.12 mg / L) to a lower level. -1 Ca + 1.08 mg·L -1 B) decreased from 68.9% to 45.6%. However, when B in the culture medium... 3+ The concentration was increased to 4.32 mg·L⁻¹. -1 Time (for MS medium B) 3+ (4 times the concentration), STN rate up to 100%; continue to increase B 3+ Concentration up to 6.48 mg·L -1 8.64 mg·L -1 At this time, the culture gradually died, showing no signs of bud proliferation, and the incidence and proliferation coefficient of STN lost statistical significance; furthermore, if Figure 5 As shown on the right Y-axis, when Ca in the culture medium 2+ Or B 3+ When the content is high, the bud proliferation coefficient will decrease; when the Ca content in the culture medium is high... 2+ B 3+ Normal concentration (120.12 mg·L⁻¹) -1 Ca 2+ + 1.08 mg·L -1 B 3+ A high proliferation coefficient (11.73) was recorded for Ca; while low concentrations of Ca... 2+ (240.24 mg·L) -1 Although a high proliferation coefficient (9.7) can be obtained, the regenerated shoots show obvious morphological degeneration, such as leaf shrinkage, stunted growth, and weakness, and are accompanied by high-frequency STN (see Figure 4 A); Continue to increase Ca 2+ Concentration up to 480.48 mg·L -1 At that time, a high proliferation coefficient (10.02) was obtained, and the height and leaves of the regenerated shoots were normal (see...). Figure 4 B); Ca 2+ The concentration increased to 720.72 mg·L⁻¹ -1 At that time, the proliferation coefficient decreased significantly, and the number of regenerated shoots decreased significantly (see...). Figure 4 C); when Ca 2+ When the concentration is too high (960.96 mg·L⁻¹), -1 Ca 2+ The proliferation coefficient was only 1.42, and the number of regenerated shoots was significantly reduced (see...). Figure 4 D). With low Ca2+ The concentration situation is similar, with lower B... 3+ Concentration (2.16 mg·L) -1 Although a high proliferation coefficient (9.63) can be obtained, the regenerated shoots also have characteristics such as leaf shrinkage, stunted growth, and weakness (see...). Figure 4 E); B 3+ The concentration was increased to 4.32 mg·L. -1 At that time, the proliferation coefficient was as low as 0.62 (see...) Figure 4 F); at a higher B 3+ At concentrations (6.48, 8.64 mg·L⁻¹) -1 The plant develops dark brown callus-like tissue at its base, but no new shoots appear, and it gradually withers and dies (see...). Figure 4 G-4H).

[0032] Example 4: Optimized culture, rooting, and acclimatization transplantation of test-tube seedlings The stem segments with nodes were inoculated into culture medium B for culture, wherein culture medium B used MS medium as the basal medium and the Ca was adjusted. 2+ The total concentration was 360.36 mg·L⁻¹. -1 Add 3.0 mg / L 6-BA and 2.0 mg / L -1 KT, 0.5 mg·L -1 NAA+; at 25 ± 1℃, light intensity of 1500-1800 lx, 7-9 hours of light per day, observe and record explant growth every 5 days (see...). Figure 6 ).

[0033] like Figure 6 As shown (scale bar = 2.0 cm), where, Figure 6 As can be seen in Figure A, after 10 days, the base of the stem segment begins to swell and dormant buds begin to sprout. Figure 6 As can be seen in B, after 20 days of cultivation, the axillary buds elongated significantly and began to show a clustered bud appearance; Figure 6 As can be seen from C, after 30 days of cultivation, the shoot clusters grew rapidly and no STN occurred; Figure 6 As can be seen from D, after 40 days of cultivation, the plants were robust, with full and unfolded leaves, thick main stems, and the incidence of STN remained below 10%.

[0034] Healthy, STN-free shoots were transferred from the optimized shoot proliferation medium to medium C for induction of rooting. Medium C used MS medium as the basal medium, supplemented with 0.5 mg / L naphthaleneacetic acid. The culture was conducted at 25 ± 1℃, with a light intensity of 1500-1800 lx and a daily light intensity of 7-9 h. After 20 days of culture in the rooting medium, when the roots reached approximately 5-7 cm in length, the plantlets were first placed indoors for 5 days to harden off, then transferred outdoors for another 10 days. After hardening off, the culture bottles were opened, and the rooted plantlets were rinsed under tap water to remove the culture medium from the roots and then soaked in a 0.1% carbendazim solution (w / v) for 5 min. After soaking, the rooted seedlings were transplanted into plastic pots filled with sterilized soil substrate (diameter: 8.0 cm; height: 8.5 cm; with drainage holes at the bottom). The pots were thoroughly watered and placed in a greenhouse for cultivation. The soil substrate consisted of coconut coir, humus, and perlite mixed in a 2:2:1 (v / v) ratio. Plant growth was observed and recorded every 5 days (see...). Figure 7 ).

[0035] like Figure 7 As shown, where, Figure 7 As can be seen from E, after 15 days of cultivation, the rooting rate was 100%, the plant growth was strong, the number of roots was large, and the rooting effect was good. Figure 7 As can be seen from F-7G, the rooted plants responded well to the soil substrate. 20 days after transplanting, the plants grew normally, with bright green leaves and well-developed root systems. Figure 7 As can be seen from H, 40 days after transplanting, the plants are robust and well-developed, with full and thick leaves, dark green color, and a survival rate as high as 95%; 4 months after transplanting, the plant stems are thick and the leaves are large.

[0036] Technical principle of the invention: (1) This invention confirms the feasibility and superiority of using segmented stems of Trichosanthes kirilowii as explants for multi-bud induction. However, it was also found that after multi-segmented explants of Trichosanthes kirilowii were inoculated into the bud propagation medium, the lower basal stem nodes swelled and produced a large number of clustered buds, while the growth of axillary buds at the upper nodes was inhibited. As the clustered buds at the base developed, they gradually changed from dormancy to withering and necrosis. This may be because there are differences in the endogenous hormone levels of each axillary bud of the multi-segmented explant. Under the stimulation of exogenous hormones, the most sensitive axillary buds at the bottom responded first and began to swell from the base of the stem, and the dormant buds sprouted one after another. The swelling of the base of the stem and the occurrence of basal buds indirectly caused damage to the vascular system of the main bud, blocking the nutrient transport path of the main bud. As a result, the uppermost axillary buds not only had their apical dominance inhibited or even lost, but were also in a state of nutrient deficiency for a long time, and their growth and development were severely inhibited, eventually withering and dying. (2) The present invention found that the establishment of an efficient micropropagation system usually depends not only on suitable explants, but also on the correct selection of plant growth regulators and their optimal concentrations. In the direct organogenesis pathway using segmented stems as materials, 6-BA is the dominant factor in inducing multi-bud regeneration, while KT and NAA play a synergistic role. The combined use of the three can better enhance the microbud proliferation performance of Trichosanthes kirilowii. The culture effect produced by the combined use of hormones is significantly higher than that produced by using them alone. This indicates that the synergistic effect between multiple hormones is far greater than the effect of their individual effects. (3) This invention confirms that the base of the plant is composed of Ca 2+ The accumulated callus tissue may indirectly cause calcium deficiency in the upper part of the plant, leading to STN or inhibiting root formation, preventing the plant from absorbing nutrients from the exogenous culture medium and synthesizing endogenous cytokinins through the roots, thus indirectly leading to STN; and it was found that during bud induction, plants with roots often did not develop STN compared to plants without roots. (4) This invention discovered that the occurrence of STN in Trichosanthes kirilowii leaves is related to the Ca in the culture medium. 2+ B 3+ Concentration-dependent. As the Ca in the culture medium... 2+ Concentration from 120.12 mg·L -1 Increased to 480.48 mg·L -1 It can effectively reduce the incidence of STN, but the higher calcium content... 2+ Concentration (960.96 mg·L) -1 Not only does it fail to completely eliminate STN, it also inhibits plant proliferation. Using CaCl₂·2H₂O as a calcium source, at higher Ca... 2+ At what concentration, Cl in MS medium -1 The concentration will also increase, and due to the toxicity of chloride itself, this will lead to yellowing of plant leaves, weak stems, and reduced proliferation rate; increasing B... 3+ Increasing Ca2+ has a less significant effect on reducing STN in Trichosanthes kirilowii leaves. 2+ This can lead to severe inhibition of culture growth, possibly due to B. 3+ The concentration difference between deficiency and toxicity is small, with minor variations in B. 3+ At higher concentrations, the concentration reaches toxic levels for plants, thus exhibiting a severe inhibitory effect on plant growth.

[0037] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the foregoing embodiments, or make equivalent substitutions for some of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for improving the efficiency of artificial propagation of Trichosanthes kirilowii, characterized in that, Includes the following steps: (1) Obtaining explants: Select healthy plants with good growth and no pests or diseases, and take stem segments with nodes; (2) Disinfect the stem segments with nodes from step (1) and cut them into appropriate sizes; (3) The segmented stem that has been disinfected and sterilized in step (2) is inoculated into culture medium A for culture to obtain sterile test-tube seedlings. Culture medium A is based on MS medium, with 0.5-1.5 mg / L 6-benzylpurine, 30000 mg / L sucrose and 4700 mg / L agar powder added. (4) Development and proliferation of basal shoot clusters: After cutting the sterile test-tube seedlings cultured in step (3) into appropriate sizes, they were transferred to culture medium B for further culture to obtain clustered shoots. Culture medium B was based on MS medium with adjusted total calcium and boron concentrations, and contained 6-benzylpurine, kinetin, and naphthaleneacetic acid. The concentration of 6-benzylpurine was 2.5-3.5 mg / L, the concentration of kinetin was 1.5-2.5 mg / L, and the concentration of naphthaleneacetic acid was 0.2-0.6 mg / L. 2+ The total concentration was 360-380 mg / L, and the total boron concentration (calculated as B) was 1.08 mg / L. (5) Take the strong cluster of buds without bud tip necrosis from step (4), and inoculate them individually in culture medium C to induce rooting. Culture medium C uses MS medium as the basic medium and adds 0.5-1.0 mg / L naphthaleneacetic acid. (6) Hardening off and transplanting: Take the rooted plants from step (5) and harden them off at room temperature. Take the seedlings out of culture medium C, clean off the residual culture medium, and disinfect them in carbendazim solution. (7) Transplant the disinfected rooted seedlings into the disinfected soil substrate and keep them warm and moist to obtain transplanted seedlings.

2. The method for improving the artificial propagation efficiency of Trichosanthes kirilowii according to claim 1, characterized in that, In step (1), the length of the stem segment with nodes is 4-5cm, and the number of nodes in the stem segment is >2.

3. The method for improving the artificial propagation efficiency of Trichosanthes kirilowii according to claim 1, characterized in that, The method for disinfecting the stem segments with nodes in step (2) is as follows: rinse with running water for 4 hours, place on a clean bench and treat with 75% ethanol solution for 5-10 seconds, then sterilize with 0.1% mercuric chloride solution for 10-12 minutes, and finally rinse with sterile water 2-3 times, each time for no less than 2 minutes.

4. The method for improving the artificial propagation efficiency of *Trichosanthes kirilowii* according to claim 1 or 2, characterized in that, The environmental conditions in steps (3)-(6) are 25 ± 1℃, light intensity of 1500-1800 lx, and 7-9 hours of light per day.

5. The method for improving the artificial propagation efficiency of *Trichosanthes kirilowii* according to claim 1 or 2, characterized in that, The environmental conditions in step (7) are 20-28℃, humidity of 60%-80%, and light intensity of 2200-2600 lx.

6. The method for improving the artificial propagation efficiency of Trichosanthes kirilowii according to claim 3, characterized in that, The pH value of the culture medium in steps (3)-(6) is 5.6-5.

8.

7. The method for improving the artificial propagation efficiency of Trichosanthes kirilowii according to claim 3, characterized in that, Before transferring the sterile test-tube seedlings into culture medium B in step (4), cut them into stem segments with a length of 4-5 cm and a number of nodes > 2.

8. The method for improving the artificial propagation efficiency of Trichosanthes kirilowii according to claim 1, characterized in that: The concentration of 6-benzylpurine in culture medium B was 3.0 mg / L, the concentration of kinetin was 2.0 mg / L, the concentration of naphthaleneacetic acid was 0.5 mg / L, and the concentration of Ca... 2+ The total concentration was 360.36 mg / L.

9. The method for improving the artificial propagation efficiency of Trichosanthes kirilowii according to claim 1, characterized in that, (6) The mass concentration of the carbendazim solution mentioned in the step is 0.1-0.2%.

10. The method for improving the artificial propagation efficiency of *Trichosanthes kirilowii* according to claim 1, characterized in that, The soil matrix is ​​made by mixing coconut coir, humus and perlite in a ratio of 2:2:1 (v / v).

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