Efficient and cost-saving seedling cultivation method for curculigo orchioides

Abstract

The present application relates to a kind of efficient and economical Curculigo orchioides seedling cultivation methods, comprising: raw material collection and processing;Curculigo orchioides leaf emergence and sprouting;Curculigo orchioides leaf culture.The leaf cutting technology used in the present application solves the problems of high production cost, high level of facilities and technical personnel of Curculigo orchioides tissue culture seedlings;At the same time, it also solves the problems of low propagation coefficient, uneconomical material selection and difficult seed propagation of Curculigo orchioides rhizome propagation;The present application collects material during the growth period of Curculigo orchioides, which can be collected, cultivated and planted on site, is efficient and economical, does not require complex facilities and equipment, does not need to go through the three processes of "callus induction-bud induction- rooting" of conventional tissue culture, has a short seedling period, and can germinate in 90 days, which is of great significance for the large-scale artificial cultivation of Curculigo orchioides.

Description

Technical Field

[0001] This invention relates to the field of Curculigo orchioides seedling production technology, and in particular to a highly efficient and cost-effective method for cultivating Curculigo orchioides seedlings. Background Technology

[0002] *Curculigo orchioides* Gaertn., a perennial herb belonging to the genus *Curculigo* in the family Amaryllidaceae, is mainly produced in Zhejiang, Jiangxi, Fujian, Taiwan, Hunan, Guangdong, Guangxi, southern Sichuan, Yunnan, and Guizhou. *Curculigo orchioides* is frequently used as a traditional medicine in China, India, and Southeast Asia, primarily for weakness in the limbs, lumbar and knee arthritis, and watery diarrhea. It can also be used as an effective immunomodulator and aphrodisiac. According to the *Chinese Pharmacopoeia* (2020 edition, Part I), *Curculigo orchioides* is used medicinally as its dried rhizome. Its main medicinal component is curculigoside, with a content not less than 0.1%. *Curculigo orchioides* is pungent, hot, and toxic; it enters the kidney, liver, and spleen meridians, tonifying kidney yang, strengthening tendons and bones, and dispelling cold and dampness. It is used for impotence, premature ejaculation, weakness of tendons and bones, cold pain in the lower back and knees, and diarrhea due to yang deficiency.

[0003] Due to the difficulty in obtaining Curculigo orchioides seeds, the difficulty in seed germination, and the long seedling cycle, seed propagation is not feasible in production. Currently, Curculigo orchioides seedling production mainly relies on rhizome propagation or tissue culture. Rhizome propagation requires a large amount of medicinal organs (rhizome) as propagation material, resulting in a low propagation coefficient, being uneconomical in terms of materials, and being time-consuming. Using Curculigo orchioides leaves or rhizomes as materials, plant in vitro rapid propagation technology is the best choice for producing Curculigo orchioides seedlings because it is fast and efficient, and can produce a large number of seedlings in a short period of time. Current technology generally involves a three-stage culture process, namely, "callus induction - bud differentiation - rooting". Factors such as explant type, location, hormone level, and culture conditions all affect the rapid propagation effect. Plant in vitro rapid propagation requires sterile conditions, specialized plant factory seedling workshops, and professional technicians, resulting in high input and production costs. Therefore, the existing Curculigo orchioides seedling system needs further improvement and optimization. Developing a highly efficient and cost-effective Curculigo orchioides seedling system is of great significance for the large-scale artificial cultivation of Curculigo orchioides. Summary of the Invention

[0004] To address the aforementioned problems, this invention proposes a highly efficient and cost-effective method for cultivating Curculigo orchioides seedlings, which allows for leaf propagation using Curculigo orchioides leaves, significantly reducing seedling costs. The specific technical solution of this invention is as follows:

[0005] A highly efficient and cost-effective method for cultivating and propagating Curculigo orchioides seedlings includes:

[0006] Step (1) Raw material collection and processing

[0007] Collect mature green leaves from healthy Curculigo plants, wash away the soil and other debris from the surface of the explant with tap water, cut off the top and narrower parts of the leaves, and cut the remaining parts into small sections.

[0008] Step (2) Inducing buds from Curculigo leaves

[0009] Using vermiculite as the culture medium, after soaking in boiling water or disinfecting with 800-1000 times diluted carbendazim solution, dispense it into hydroponic containers, add water to ensure that it just covers the surface of the substrate, place the leaf segments flat on the substrate surface, and place them in a culture room or at room temperature with diffused light for culture. During this period, observe the growth of the buds and replenish water in time to ensure that the substrate is just submerged in water.

[0010] Step (3) Cultivation of robust seedlings of Curculigo orchioides

[0011] After the leaves sprout, add 1 / 4 MS nutrient solution or general nutrient solution for soilless cultivation that does not contain organic matter and sucrose to promote strong seedling growth. During this period, replenish water to keep the substrate moist. Later, open the lid and continue to cultivate for a period of time to achieve the dual effect of strengthening and hardening the seedlings. When most of the seedlings are about 7cm tall and the roots are 4-6cm long, they can be transplanted.

[0012] Furthermore, in step (1), the two ends of the leaves of the Curculigo orchioides should be cut off, and the remaining part should be cut into small sections of about 3cm.

[0013] Furthermore, in step (1), the collection time is from June to September, and different regions need to make arrangements according to the phenological period of Curculigo orchioides.

[0014] Furthermore, in step (2), vermiculite is selected as the substrate, with a thickness of 4-5cm. The substrate treatment method is: soaking in boiling water for 5-10 minutes or soaking in 800-1000 times diluted carbendazim solution for 5-10 minutes before use.

[0015] Furthermore, in step (2), only water is used during the budding stage. The plants are cultured under ambient temperature diffused light or in a culture room with the following conditions: 10-14 hours of light, 1000-2000 lx, and 26±0.5℃. During this period, the water level is just above the substrate to ensure that the leaves can absorb water. No nutrient solution or hormones are added before budding.

[0016] Furthermore, in step (3), from the start of leaf cutting to 40-50 days after budding, nutrients need to be supplemented to ensure that the buds can develop normally into seedlings. At this time, add 1 / 4 MS or general nutrient solution for soilless cultivation that does not contain organic matter and sugar.

[0017] Furthermore, in step (3), 60-70 days after the leaf cutting begins, the seedlings have fully developed. At this time, opening the seedling cover and continuing cultivation can achieve the dual effect of strengthening the seedlings and hardening them. During this period, water should be added to keep the substrate moist. When most of the seedlings are about 7cm tall and the roots of the seedlings are 4-6cm long, they can be transplanted.

[0018] The present invention also relates to the *Curculigo orchioides* obtained by the above-described method.

[0019] This invention is based on an accidental discovery during experiments (budding of Curculigo leaves placed in a bottle). During the development process, initially, conventional MS and 1 / 2 MS solutions were used as the basic nutrient solutions, with different hormones added to promote leaf healing and budding. However, because the induction process was conducted under light in a bacterial environment, mold growth appeared on the leaves, and algae grew in the substrate. Later, the leaves turned brown and died, significantly reducing the budding rate. Later, orthogonal experiments revealed that water without any added nutrient solution or hormones was the most effective for inducing budding. However, if the entire process... Without nutrient solution, seedlings may develop browning or necrosis at the tips due to insufficient nutrition. Adding nutrient solution containing sugar and organic matter can also lead to a deterioration of the substrate environment and mold growth. The timing and type of nutrient solution added are very important. Adding it too early, before the buds have developed properly, can lead to excessive algae growth, which will inhibit the development of the seedlings. After repeated trials, it was found that adding 1 / 4 MS solution without organic matter and sugar after 40-50 days of cultivation can promote the development of seedlings. In small-scale trials, a general-purpose nutrient solution for soilless cultivation was used to reduce costs and satisfactory results were also achieved.

[0020] The leaf cutting technique used in this invention solves the problems of high production costs, high requirements for facilities and equipment, and high skill levels of technicians required for tissue culture seedlings of Curculigo orchioides. It also solves the problems of low propagation coefficient, uneconomical material sourcing, and difficulty in seed propagation of Curculigo orchioides. The seedling cultivation technology of this invention can collect materials during the growing season of Curculigo orchioides, from June to September, and cultivate and plant seedlings on-site. It is highly efficient and cost-effective, does not require complex facilities and equipment, and does not go through the three processes of "callus induction - bud differentiation - rooting" of conventional tissue culture. The seedling cycle is short, with seedlings emerging in 90 days, which is of great significance for the large-scale artificial cultivation of Curculigo orchioides.

[0021] Currently, the market price of tissue culture seedlings used in production is 0.4 yuan per seedling. Based on the current requirement of 30,000 seedlings per mu for understory cultivation, the cost of seedlings for farmers is 12,000 yuan per mu. The cost of agricultural materials and other inputs is calculated at 1,000 yuan per mu per year. It takes 3 years after planting for Curculigo orchioides to be harvested, so the investment cost per mu is 15,000 yuan. At present, the yield of dried Curculigo orchioides is 126 kg per mu, the price is 140 yuan per kg, the output value is 17,640 yuan per mu, and the farmer's benefit is only 2,640 yuan per mu.

[0022] The Curculigo orchioides leaf propagation technique of this invention allows for the use of locally sourced materials. If two leaves are taken from each plant for propagation, each leaf segment can conservatively be cut into five segments, resulting in ten leaf segments per plant and 300,000 leaf segments per acre. Using this seedling cultivation technique, the germination rate is conservatively estimated at 70%, and the transplant survival rate at 80%. Each acre of cultivation base can produce 168,000 effective seedlings from Curculigo orchioides leaves, sufficient for a 5.6-acre base. The only required facilities are a shade structure and a hydroponic pond, a one-time investment. The hydroponic pond can be constructed with hollow bricks and lined with thick plastic sheeting to reduce costs. According to the method of this invention, if the leaves are not overlapped, approximately 1600 leaf segments can be placed per square meter. During seedling cultivation, the vermiculite thickness is calculated at 5cm, requiring 0.05m³ of substrate per square meter. 3 The price of vermiculite is 1200 yuan per cubic meter, which can produce 20m³ of vermiculite. 2 For the seedbed, 1 / 4 MS nutrient solution can be replaced with a general nutrient solution for hydroponics, per 1m 3 The nutrient solution costs 35 yuan per cubic meter for 20m³ of water and fertilizer. 2 The initial investment in vermiculite and nutrient solution for the seedbed was 1235 yuan, averaging only 62 yuan per square meter. Based on a 70% germination rate, each square meter could produce 1120 seedlings, with a cost of approximately 0.06 yuan per seedling. With a cultivation rate of 30,000 seedlings per mu (approximately 0.067 hectares), the cost of seedlings was 1800 yuan per mu, resulting in a cost reduction of 10,200 yuan and significantly increasing farmers' profits.

[0023] This invention has low production costs, requiring only simple seedling facilities. Mature leaves of Curculigo orchioides can be collected at the cultivation base. Consumables such as vermiculite, tap water, low-concentration 1 / 4MS or general nutrient solution for soilless cultivation are required. Compared with plant tissue culture seedling cultivation, which requires a large investment in facilities, equipment and culture media, the cost is greatly reduced.

[0024] The production process of this invention is simple to operate and does not require complicated aseptic operations and indoor controlled cultivation processes. Even farmers without knowledge background can quickly get started to produce Curculigo seedlings after training. Conventional plant tissue culture requires aseptic conditions and has higher technical requirements for seedling personnel.

[0025] This invention has a high propagation coefficient and low variation. At least 5 seedlings can be obtained from one normal mature leaf. No plant hormones need to be added. Compared with the addition of various hormones in the process of plant tissue culture, the probability of gene mutation is greatly reduced by the method of seedling formation through dedifferentiation and redifferentiation.

[0026] This invention features a short seedling cultivation cycle, yielding transplantable seedlings in just 90 days, 20 days earlier than the existing technology CN201810969410.5. Furthermore, since the seedlings are cultivated under natural conditions, a hardening-off process has been incorporated into the cultivation process. The seedlings are hardened off after 60 days of cultivation by opening the lid, and transplanting can be done directly after 90 days. The optimal combination scheme can achieve a survival rate of over 90%.

[0027] This invention can not only be used to produce seedlings, but also effectively protect wild resources and promote the preservation of wild germplasm resources. Wild resources can be collected without digging up the whole plant, but by taking leaves and using the method of this invention for propagation and preservation. This preserves the genetic characteristics of the resources and does not damage the resources in their original habitat, thus having good social and ecological benefits. Attached Figure Description

[0028] Figure 1 This is a phenomenon of bud break from the midrib tissue of a *Curculigo orchioides* leaf segment according to an embodiment of the present invention; wherein: a. early healing stage; b. mid-bud break stage; c. longitudinal section micrograph of bud break from a *Curculigo orchioides* leaf segment;

[0029] Figure 2 These are microscopic sections of the midrib and bud of Curculigo orchioides leaves according to embodiments of the present invention; wherein, a. cross section of the midrib of Curculigo orchioides leaf (before differentiation); b. early section of Curculigo orchioides leaf cuttings;

[0030] Figure 3 This is a flowchart illustrating the method operation of an embodiment of the present invention;

[0031] Figure 4 These are images showing the effects of different stages of Curculigo orchioides leaf cuttings according to an embodiment of the present invention; wherein: a. 30d; b. 60d; c. 90d;

[0032] Figure 5 These are illustrations of the cultivation effect of Curculigo orchioides leaf cuttings according to an embodiment of the present invention; wherein: a. before transplanting; b. 30 days after transplanting; c. 6 months after transplanting; d. 1 year after transplanting. Detailed Implementation

[0033] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention. Where specific techniques or conditions are not specified in the embodiments, they are performed according to the techniques or conditions described in the literature in the art or according to the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased.

[0034] Unless otherwise stated, all percentages in this invention represent mass fractions. Ratios are mass percentages, and concentrations are mass concentrations.

[0035] In our experiments, we accidentally discovered that when wild Curculigo leaves are soaked in water, callus tissue forms at the midrib of the cut end at the bottom of the leaf, and then buds differentiate from it. Figure 1 As shown, the callus tissue of *Curculigo orchioides* originates from the central midrib, and roots and buds differentiate from the callus tissue, indicating that *Curculigo orchioides* leaves have strong regenerative and photosynthetic autotrophic capabilities. Freehand sections of the leaves and bud locations of *Curculigo orchioides* were prepared and observed, with results as follows: Figure 2 As shown in Figure a, the periphery of the vascular bundles in the midrib of the Curculigo leaf contains numerous fiber cells, with a large proportion of xylem. The xylem contains many parenchyma cells, and there are 1-2 layers of smaller cells between the xylem and phloem, which may be meristem. When the callus tissue of Curculigo was transversely sectioned along the vertical direction of the leaf, the results... Figure 2 As shown in Figure b, it can be seen that vascular tissue differentiation begins in the middle cells of the callus tissue section. The middle cells are smaller and denser, belonging to typical meristematic tissue. A longitudinal section of a late-stage callus bud yields... Figure 1 As shown in Figure c, it can be seen from the figure that the callus tissue of Curculigo orchioides leaf segments differentiates into buds in an organogenesis-type manner. Most callus first differentiates into roots and then into buds. Therefore, it is speculated that leaf cuttings can be used to quickly propagate Curculigo orchioides.

[0036] Example 1

[0037] This embodiment of the efficient and cost-saving method for cultivating and propagating Curculigo orchioides seedlings uses wild Curculigo orchioides leaves from the wild Curculigo orchioides germplasm resource nursery in Boshan Town, Linxiang District, Lincang City as materials. It is the first time that the leaf cutting method has been used to study the rapid propagation system of Curculigo orchioides, providing technical support for the efficient and cost-saving propagation of Curculigo orchioides seedlings.

[0038] like Figure 3 As shown in the figure, leaves from healthy wild Curculigo orchioides germplasm resources in Boshan Town, Linxiang District, Lincang City were collected as explants in this embodiment.

[0039] Step (1) Raw material collection and processing

[0040] Collect mature green leaves from healthy Curculigo plants, wash away soil and other debris from the surface of the explant with tap water, cut off the top and narrower parts of the leaves, and cut the remaining parts into small sections of about 3cm.

[0041] Step (2) Curculigo leaves emerge and buds appear

[0042] Using the basic nutrient solution (without added organic matter and sucrose), NAA, and 6-BA concentrations as experimental factors, the L9(3) method was employed. 4Nutrient solution formulations were designed using an orthogonal array with a pH of 5.8. The formulations are shown in Table 1. Vermiculite was used as the culture medium. After soaking in boiling water for 10 minutes, the medium was dispensed into seedling trays with a substrate thickness of approximately 3 cm. The nutrient solution shown in Table 2-1 was added, with the volume just covering the substrate surface. Leaf segments were inoculated into the medium with the lower epidermis facing down. Each formulation was used to inoculate 3 trays, with 12 leaf segments inoculated per tray. The trays were then placed in a culture room (light, 12h, 2000lx) at a temperature of 26±0.5℃. After 60 days of culture, the germination rate and algae contamination rate were recorded. During this period, the growth of the buds was observed, and water was replenished as needed to ensure the substrate was completely submerged in the nutrient solution.

[0043] Table 1. Nutrient solution formula for induction culture of Curculigo buds

[0044] formula Basic nutrient solution NAA (mg / L) 6-BA (mg / L) XMY1 water 0 0 XMY2 water 0.1 0.2 XMY3 water 0.2 0.4 XMY4 1 / 2MS 0 0.2 XMY5 1 / 2MS 0.1 0.4 XMY6 1 / 2MS 0.2 0 XMY7 1 / 4MS 0 0.4 XMY8 1 / 4MS 0.1 0 XMY9 1 / 4MS 0.2 0.2

[0045] Step (3) Cultivation of Curculigo leaves

[0046] After selecting the optimal nutrient solution formula, the best bud induction nutrient solution was chosen to study the effects of substrate, leaf segment length, and inoculation method on bud induction and seedling growth of Curculigo orchioides. The experimental protocol is shown in Table 2. The culture substrate was filled into seedling boxes with a size of 165mm×125mm according to the experimental formula, with a substrate thickness of 4-5cm. Water was added, just enough to cover the substrate surface. Leaf segments of the corresponding size were inoculated according to the experimental formula, with 12 leaf segments inoculated per box. Each treatment was replicated 3 times. The boxes were placed in a culture room for 12 hours at 2000lx at a temperature of 26±0.5℃. After 45 days of culture, 1 / 4 MS (excluding organic matter and sucrose) nutrient solution was added to each treatment for seedling strengthening. Water was added during the process to keep the substrate moist. After 60 days of culture, the budding rate was recorded. The seedling box lid was opened, and the seedlings were cultured for another 30 days, during which time the substrate was kept moist. Ninety days after leaf cuttings, plant height, root length, and root quantity were measured. The seedlings were then transplanted into pots filled with nutrient soil. After transplanting, shading and moisture retention were maintained. The survival rate of the seedlings was observed 15 days after transplanting, and the transplant survival rate was calculated.

[0047] Table 2 Experimental protocols for the culture of Curculigo orchioides leaves with different substrates, leaf segment lengths, and inoculation methods.

[0048] formula Culture substrate Leaf segment length (cm) vaccination method JZ1 vermiculite 3 lay flat JZ2 perlite 3 lay flat JZ3 Vermiculite perlite 1:1 3 lay flat JZ4 sand 3 lay flat YC1 vermiculite 2 lay flat YC2 vermiculite 3 lay flat YC3 vermiculite 4 lay flat YC4 vermiculite 3 Vertical insertion

[0049] (Note: JZ1-JZ4 represent different substrate treatments, YC1-YC3 represent different leaf segment lengths, and YC2 and YC4 represent leaf placement methods.)

[0050] In this embodiment, Excel is used to organize and calculate the original data, SPSS 22.0 is used to process and analyze the experimental data, and Duncan's method is used for analysis of variance and multiple comparisons. Percentage data are transformed by square root arcsine before analysis of variance and multiple comparisons are performed.

[0051] After 60 days of inoculation, the germination rate of each treatment was statistically analyzed. SPSS 22.0 was used for analysis of variance and multiple comparisons, and the results are shown in Table 3. The analysis of variance results in Table 3 show that NAA and 6-BA had no significant effect on the germination rate of *Curculigo orchioides* leaves, while the basic nutrient solution had a significant effect. Duncan's method was used for significance analysis and multiple comparisons, and the results are shown in Table 4. When the basic nutrient solution was water, the germination rate of *Curculigo orchioides* was the highest (43.70%), followed by 1 / 4MS, and the germination rate of 1 / 2MS was the lowest. Table 5 shows that different nutrient solution formulations had a significant effect on the germination rate of *Curculigo orchioides* leaf segments. The results of multiple comparisons are shown in Table 6. The formulations XMY1, XMY2, XMY7, and XMY4 had higher germination rates, with XMY1 (only water added) showing the best effect; the formulations XMY3, XMY5, XMY6, XMY8, and XMY9 had lower germination rates. The above results indicate that water can be used directly during the bud induction stage of Curculigo orchioides without the need to add nutrient solution or hormones.

[0052] Table 3. Analysis of variance on the effects of basic nutrient solution, NAA, and 6-BA on the germination rate of Curculigo leaves.

[0053]

[0054] Table 4 Germination rate of Curculigo orchioides under different basic nutrient solutions

[0055] Basic nutrient solution Germination rate (%) Deionized water 43.70±7.00(39.24±5.81)a 1 / 4MS 28.15±7.00(28.47±5.81)ab 1 / 2MS 15.56±7.00(17.55±5.81)b

[0056] Note: The values ​​in parentheses are the values ​​after square root arcsine conversion. The percentage data below are processed in the same way as in this table.

[0057] Table 5. Analysis of variance of germination rate of Curculigo leaves with different formulations.

[0058]

[0059] Table 6 Comparison of germination rate of Curculigo leaves with different formulations

[0060]

[0061]

[0062] During the experiment, it was found that algae growth occurred due to the light conditions during material cultivation. The algae contamination rate was statistically analyzed for each treatment after 40 days of inoculation, as shown in Table 7. The algae contamination rate was higher when the basic nutrient solution was 1 / 2 MS and 1 / 4 MS due to the presence of various mineral ions, with the contamination rate reaching 100% when using 1 / 2 MS. No algae contamination was observed when using water. Therefore, it can be concluded that using water instead of the basic nutrient solution eliminates algae contamination and can be used for bud induction in the early stages of Curculigo leaf cuttings.

[0063] Table 7 Algae contamination rate of different nutrient solutions

[0064] Basic nutrient solution Algal contamination rate (%) 1 / 2MS 100 1 / 4MS 66.67 water 0

[0065] Table 8 shows that leaf segment length had no significant effect on the germination rate, plant height, root length, and transplant survival rate of Curculigo orchioides. Table 9 shows that the germination rate of all inoculation lengths reached over 80%, the plant height reached over 9 cm, the root length reached over 3 cm, and the transplant survival rate reached over 70%. Considering these indicators, a leaf segment length of 3 cm resulted in better plant height, root length, and transplant survival rate for Curculigo orchioides leaf cuttings, and can be considered the optimal length for sugar-free culture of Curculigo orchioides.

[0066] Table 8. Analysis of variance on the effects of different leaf segment lengths on the culture of Curculigo orchioides leaves.

[0067]

[0068]

[0069] Table 9 Comparison of leaf cuttings with different leaf segment lengths

[0070]

[0071] Table 10 shows that different culture media significantly affected the germination rate and root length of Curculigo orchioides. The results of post-hoc multiple comparisons using the Duncan method are shown in Table 11. It can be seen that vermiculite was the highest germination rate (100%), the average root length reached 6.18 cm, and the transplant survival rate (84.52%) was also highest when vermiculite was used as the substrate. In conclusion, vermiculite can be considered the optimal culture medium for sugar-free Curculigo orchioides cultivation.

[0072] Table 10. Analysis of variance on the effects of different culture media on the culture of Curculigo orchioides leaves.

[0073]

[0074] Table 11 Results of Curculigo orchioides leaf culture on different culture media

[0075]

[0076]

[0077] Table 12 shows that the p-values ​​for different inoculation methods on the germination rate of Curculigo orchid leaf segments, the plant height of leaf cuttings, and the survival rate of transplanted Curculigo orchids were all greater than or equal to 0.05, indicating that their effects were not significant. Different inoculation methods significantly affected the root length of Curculigo orchids. Multiple post-hoc comparisons showed that the root length of Curculigo orchids cultivated through horizontal inoculation was longer than that of those cultivated through vertical inoculation. In summary, Table 13 shows that the germination rate, root length, and transplant survival rate of horizontal inoculation were all higher than those of vertical inoculation, with only a slightly lower plant height. The plants exhibited well-developed root systems and were robust. Therefore, horizontal inoculation can be considered the optimal inoculation method for sugar-free Curculigo orchid cultivation.

[0078] Table 12. Analysis of variance table showing the effect of different inoculation methods on the cultivation of Curculigo orchioides.

[0079]

[0080] Table 13 Results of Curculigo leaf culture using different inoculation methods

[0081]

[0082] To verify the optimal scheme described above, wild Curculigo orchioides leaf segments were used as experimental material, vermiculite as the culture medium, and seedlings were raised in 44cm×30cm seedling boxes. The seedling boxes contained 4-5cm of vermiculite, and tap water was added just enough to cover the substrate. After cleaning and processing, Curculigo orchioides leaves were cut into 3cm segments and inoculated flat onto the vermiculite. The seedlings were cultured under diffused light under natural conditions, with water added as needed during the cultivation process. After 45 days of cultivation, general hydroponic nutrient solution was added until it just covered the substrate. After 60 days of cultivation, the seedlings were opened for further cultivation, with regular watering. After 90 days of cultivation, the germination rate was recorded. For each treatment, 30 seedlings were randomly selected to measure plant height, root length, and root mass. All the resulting seedlings were then transplanted into nutrient soil, and the survival rate was recorded after 30 days. Leaf cuttings that had been planted for nearly a year were carefully removed from the soil, and 30 were randomly selected to measure the number and length of fleshy fibrous roots, as well as the weight of the rhizome. To estimate the propagation coefficient of leaf cuttings, 50 Curculigo orchioides plants were randomly selected from the Curculigo orchioides germplasm resource nursery. The number of collectable leaves and the length of each leaf were counted. Based on the optimal leaf length (3cm) obtained in this study, the thinner parts at both ends of the leaves removed during leaf processing (totaling 5cm) were removed. The effective propagation length of the remaining leaves was calculated, and the leaf cutting propagation coefficient of Curculigo orchioides was estimated.

[0083] The sprouting effect of Curculigo orchioides in a small test is as follows Figure 4As shown, leaf cuttings begin to sprout in 1 month, leaves emerge in 2 months, and two leaves appear in 3 months. Table 14 shows the various indicators of the leaf cuttings. The germination rate after 90 days was 72%, the root length was 6.57 cm, the root quantity was 3.77 roots per plant, the plant height was 7.99 cm, and the transplant survival rate reached 93%. One year after transplanting, the plant height reached 19.20 cm, the root length reached 8.40 cm, the root quantity reached 5.10 roots, and the root weight per plant reached 2.01 g. The leaf cuttings have well-developed root systems and grow vigorously. Figure 5 As shown in Table 14, the predicted leaf propagation coefficient results show that each *Curculigo orchioides* plant can have an average of 3 leaves for leaf propagation (ensuring that cutting does not affect the growth of *Curculigo orchioides*, retaining at least half of the leaves per plant). The average length of each leaf is 20.7 cm. After deducting the thinner ends of the leaves cut off during leaf processing (totaling 5 cm), the remaining effective length is 15.7 cm. According to the results of this small-scale test, the germination rate is 72%, the transplant survival rate is 93%, and the leaf propagation coefficient per *Curculigo orchioides* plant is 10.51, meaning that each *Curculigo orchioides* plant can yield approximately 10 seedlings through leaf propagation. The leaf propagation method for *Curculigo orchioides* seedlings using this embodiment requires simple equipment, has low cost, high transplant survival rate, and produces vigorous seedling growth. Ordinary people can easily cultivate seedlings themselves after mastering this method, making it suitable for promotion and application.

[0084] Table 14 Results of a small-scale trial using Curculigo leaf cuttings

[0085]

[0086]

[0087] Example 2

[0088] The efficient and cost-saving method for cultivating and propagating Curculigo orchioides seedlings in this embodiment includes:

[0089] Step (1) Raw material collection and processing

[0090] Collect mature green leaves from healthy Curculigo plants that have grown from leaf cuttings that have been cultivated for one year. Wash away any debris from the surface of the explant with tap water, cut off the top and narrower parts of the leaves, and cut the remaining parts into small sections of about 3cm.

[0091] Step (2) Inducing buds from Curculigo leaves

[0092] Vermiculite was used as the culture medium and disinfected with 800 times carbendazim solution for 7 minutes. It was then spread in the seedling box to a thickness of about 4.5 cm. Leaf segments were laid flat and inoculated on the surface of the substrate. The seedling box was placed in a room temperature diffused light environment for cultivation. During the cultivation, the ventilation holes of the seedling box were kept open and water was added in time to ensure that the substrate was just submerged in water. After 40 days, the germination rate was 82%.

[0093] Step (3) Cultivation of robust seedlings of Curculigo orchioides

[0094] From the start of leaf cuttings to 50 days, add the general-purpose hydroponic nutrient solution developed by Yunnan Dream New Fertilizer Co., Ltd. according to the instructions to cultivate strong seedlings. During this period, replenish water to ensure the substrate is moist. 65 days after the start of leaf cuttings, the seedlings have fully developed. Open the lid and continue cultivation for 90 days. During this period, replenish water to keep the substrate moist, achieving the dual effect of strengthening and hardening the seedlings. When most seedlings are about 5-7cm tall and the roots are 4-5cm long, transplant them. The transplant survival rate is 88%.

[0095] Example 3

[0096] The efficient and cost-saving method for cultivating and propagating Curculigo orchioides seedlings in this embodiment can be used for the preservation of wild Curculigo orchioides resources, including:

[0097] Step (1) Raw material collection and processing

[0098] Mature green leaves from healthy wild Curculigo plants were collected and washed with tap water to remove debris from the explant surface. The top and narrower parts of the leaves were cut off, and the remaining parts were cut into small sections of about 3 cm. Each plant was numbered, and the material from each plant was placed in a separate seedling box for cultivation. The resulting seedlings were their clonal lines, which facilitated later identification.

[0099] Step (2) Inducing buds from Curculigo leaves

[0100] Vermiculite was used as the culture medium, with a thickness of approximately 4.5 cm. After soaking in boiling water for 8 minutes, it was divided into small seedling trays, and water was added to ensure that the substrate surface was just submerged. Leaf segments were neatly laid flat and inoculated onto the substrate surface, and the trays were placed in a culture room for cultivation. During this period, the growth of the buds was observed, and water was added as needed to ensure that the substrate was just submerged. During the bud induction stage, only water was used, and the trays were cultivated at room temperature under diffused light or in a culture room with the following conditions: 14 hours of light, 1500 lx, and 26 ± 0.5℃. During this period, the water was ensured to just submerge the substrate to ensure that the leaves could absorb water. No nutrient solution or any hormones were added before budding.

[0101] Step (3) Cultivation of robust Curculigo seedlings

[0102] From the start of leaf cuttings until 50 days after budding, add 1 / 4 MS (containing no organic matter or sugar) to promote strong seedling cultivation. During this period, replenish water to ensure the substrate remains moist. 70 days after the start of leaf cuttings, when the seedlings have fully developed, open the lid and continue cultivation for another 20 days, replenishing water to keep the substrate moist, achieving the dual effect of strengthening and hardening the seedlings. When most seedlings reach a height of about 5-7cm and the roots of the seedlings have grown to 4-5cm, they can be transplanted.

[0103] Using the method in Example 1, a small-scale trial was conducted, and the transplant survival rate reached over 90%.

[0104] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A highly efficient and cost-saving method for cultivating and propagating Curculigo orchioides seedlings, characterized in that: include: Step (1) Raw material collection and processing Collect mature green leaves from healthy Curculigo plants, wash away debris from the surface of the explant with tap water, cut off the top and narrower parts of the leaves, and cut the remaining parts into small sections. Step (2) Inducing buds from Curculigo leaves Using vermiculite as the culture medium, after soaking and sterilizing it in boiling water or 800-1000 times diluted carbendazim solution, it is divided into hydroponic containers. Water is added to ensure that the substrate surface is just covered. Leaf segments are laid flat and inoculated on the substrate surface. The container is placed in a culture room or under diffused light at room temperature. During the culture period, the growth of the buds is observed and water is added in time to ensure that the substrate is just covered by water. Step (3) Cultivation of robust seedlings of Curculigo orchioides After the leaves sprout, add 1 / 4 MS nutrient solution or general nutrient solution for soilless cultivation that does not contain organic matter and sucrose to promote strong seedling cultivation. During this period, replenish water to ensure that the substrate is moist. Later, open the lid and continue to cultivate for a period of time to achieve the dual effect of strong seedlings and hardening off the seedlings. When most of the seedlings are about 7cm tall and the roots are 4-6cm long, they can be transplanted. In step (2), vermiculite is selected as the substrate, with a thickness of 4-5cm. The substrate treatment method is: soaking in boiling water for 5-10 minutes or soaking in 800-1000 times diluted carbendazim solution for 5-10 minutes before use. In step (2), only water is used during the budding stage. The plants are cultured at room temperature under diffused light or in a culture room. The conditions are: 10-14 hours of light, 1000-2000 lx, 26±0.5℃. During this period, the water level is just above the substrate to ensure that the leaves can absorb water. No nutrient solution or hormones are added before budding.

2. The method according to claim 1, characterized in that: In step (1), the two ends of the leaves of the Curculigo orchioides should be cut off, and the remaining part should be cut into small sections of about 3cm.

3. The method according to claim 1, characterized in that: In step (1), the collection time is from June to September. Different regions need to make arrangements according to the phenological period of Curculigo orchioides.

4. The method according to claim 1, characterized in that: In step (3), it takes 40-50 days from the start of leaf cutting to the emergence of buds. After the buds emerge, nutrients need to be supplemented to ensure that the buds can develop normally into seedlings. At this time, add 1 / 4 MS or general nutrient solution for soilless cultivation that does not contain organic matter and sugar.

5. The method according to claim 1, characterized in that: In step (3), 60-70 days after leaf cutting begins, the seedlings will have fully developed. At this time, open the seedling cover and continue cultivation. During this period, replenish water to keep the substrate moist. When most of the seedlings are about 7cm tall and the roots of the seedlings are 4-6cm long, they can be transplanted.

6. The Curculigo orchioides obtained by the method according to any one of claims 1-5.

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