A method for preserving and commercialized rapid propagation of green flowered tree fern germplasm
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- YUNNAN FENGHE SHULI BIOTECHNOLOGY CO LTD
- Filing Date
- 2025-06-30
- Publication Date
- 2026-06-23
Smart Images
Figure CN120615720B_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the field of plant tissue culture and propagation technology, specifically to a method for the preservation and rapid commercial propagation of *Echeveria elegans* germplasm. Background Technology
[0002] The statements in this section provide only background information relevant to the disclosure of this application and may not constitute prior art.
[0003] Green-flowered Stone Lotus ( Sinocrassula indica var. viridiflora KT Fu is a member of the Crassulaceae family and the Echeveria genus. Sinocrassula *Echeveria elegans* is a perennial herbaceous plant mainly distributed in limestone areas at altitudes of 1500-3000 m in central and northern Yunnan, including Dali, Lijiang, and Kunming. It grows primarily in rock crevices or steep cliffs, with populations distributed in patches, indicating a unique and fragile habitat. According to the latest survey, *Echeveria elegans* is scarce and easily threatened by invasive species, with its wild population already classified as Vulnerable (VU). *Echeveria elegans* possesses significant ecological and ornamental value; its unique rosette-shaped plant form and gray-green leaves are highly ornamental, making it a rare and valuable material for alpine succulent landscaping. The leaves of *Echeveria elegans* are rich in flavonoids, exhibiting significant antioxidant activity, and its potential medicinal value warrants further development. Furthermore, as a pioneer plant in limestone areas, *Echeveria elegans* plays an important role in combating desertification, as its roots secrete organic acids that dissolve rocks and promote soil formation.
[0004] Succulents reproduce naturally through both sexual and asexual means. While sexual reproduction can produce variations that are beneficial for evolution and adaptation, its seed germination rate is low and the cycle is long, requiring 2-3 years or even longer to flower. Vegetative propagation, such as through bulbils, runners, and leaves, can maintain the characteristics of the parent plant, but its propagation coefficient is limited and it is highly susceptible to pests and diseases. Artificial propagation methods for succulents mainly include cuttings, grafting, and tissue culture. Cuttings are simple to perform and have a survival rate of around 90%, but are limited by season and have a limited propagation coefficient. Grafting can quickly obtain superior traits, but it requires high skill and is prone to infection at the graft union. Tissue culture allows for large-scale, rapid propagation, with an annual propagation coefficient reaching 10. 5 -10 6 This demonstrates its broad prospects in the commercial application of succulent plants. In recent years, significant breakthroughs have been made in the in vitro culture technology of succulent plants, mainly focusing on explant selection, regeneration system optimization, and culture environment control, achieving some results; however, most studies still rely on TDZ-induced somatic embryo formation, resulting in problems such as high cost, a large proportion of deformed seedlings, and low reproducibility.
[0005] In the in vitro rapid propagation of succulents, there are three main ways to obtain regenerated plants: (1) direct organogenesis, where adventitious buds are directly differentiated from explants such as leaves, without going through the callus stage; (2) indirect organogenesis, where explants such as leaves and inflorescences first form callus, and then plant growth regulators are used to induce the callus to differentiate into adventitious buds and roots; (3) somatic embryogenesis, which includes two modes: direct somatic embryogenesis—somatic cells directly differentiate into embryos under in vitro culture conditions, without going through the callus stage; and indirect somatic embryogenesis—somatic cells first dedifferentiate into callus, and then some cells in the callus redifferentiate into embryos. Among these three pathways, direct organogenesis does not go through the callus stage and directly differentiates buds or roots from explants, which can shorten the culture cycle and reduce the probability of contamination during long-term callus culture. However, it is only suitable for succulents with strong regeneration ability and has low efficiency for species that are difficult to regenerate. It requires the selection of young and healthy explants; the regeneration ability of old leaves or damaged tissues is significantly reduced. Indirect organogenesis has broad applicability, overcoming the limitations of direct explant regeneration through the callus stage. It is suitable for succulents with difficult regeneration, and the callus can be preserved for a long time and proliferate in large quantities, making it suitable for commercial production. However, it also has the problem of a long culture cycle. Somatic embryogenesis has high regeneration efficiency, but requires strict control of hormones and culture conditions; otherwise, problems such as deformed embryos or developmental arrest are likely to occur.
[0006] The prior art document with publication number CN106613988B discloses a method for rapid cultivation of commercial small potted plants of the genus Echeveria that are difficult to propagate by cuttings using bottle molding. Leaf tissue containing axillary bud meristems at the leaf base is used as an aseptic explant. Method (1) direct organogenesis pathway is adopted. Induction medium (MS + ZT 1.0~2.0 mg / L + NAA 0.1~0.2 mg / L + adenine sulfate 30~50 mg / L + sucrose 25~30 g / L + agar 6.0~7.0 g / L, pH value 5.5~6.0) is used to directly induce shoot clusters without going through the callus stage. The induced shoot clusters are used in proliferation medium (MS + ZT 0.2~0.3 mg / L + NAA 0.1~0.2 mg / L + adenine sulfate 30~50 mg / L + sucrose 25~30 mg / L) Proliferation was induced using a medium containing 300–500 mg / L activated charcoal and 6.0–7.0 g / L agar, pH 5.5–6.0; subsequently, rooting induction was performed using a seedling-strengthening and rooting medium (1 / 2 MS + 0.3–0.5 mg / L NAA + 25–30 g / L sucrose + 300–500 mg / L activated charcoal + 6.0–7.0 g / L agar, pH 5.5–6.0). This existing technology uses leaf tissue containing axillary bud meristems at the leaf base as explants and employs a combination of induction media. It bypasses the callus stage, resulting in a low propagation coefficient and a limited number of seedlings obtained within the cycle. Summary of the Invention
[0007] The purpose of this invention is to address the problems existing in current technologies by providing a method for the germplasm preservation and rapid commercial propagation of *Echeveria elegans*. This method involves establishing a sterile culture system using mature leaves as explants, optimizing the effects of embryogenic callus induction medium, somatic embryogenesis medium, and plant regeneration medium, successfully inducing embryogenic callus and achieving efficient proliferation, thereby establishing a stable and efficient regeneration system. The research results have both urgent and ecological economic value for the germplasm resource conservation and sustainable commercial utilization of this high-value endemic plant, *Echeveria elegans*.
[0008] The technical solution of the present invention is as follows:
[0009] A method for germplasm preservation and rapid commercial propagation of Echeveria 'Green Flower' includes the following steps:
[0010] Step S1: Embryogenic callus proliferation: Explant material was obtained from sterile seedlings and inoculated into embryogenic callus proliferation medium to induce embryogenic callus.
[0011] The culture medium for the proliferation of embryogenic callus is: MS + 1.0 mg / L-2.0 mg / L 6-BA + 0.5 mg / L-1.0 mg / L NAA or MS + 1.0 mg / L-2.0 mg / L 6-BA + 0.5 mg / L-1.0 mg / L 2,4-D;
[0012] Step S2: Proliferation of callus-somatic embryo mixture: The embryogenic callus tissue obtained in step S1 is further cultured until a callus-somatic embryo mixture is formed. The obtained callus-somatic embryo mixture is cut into small pieces suitable for inoculation and inoculated into the callus-somatic embryo mixture proliferation medium for proliferation induction to obtain clustered shoots.
[0013] Step S3: Divide the clustered shoots induced in step S2 into individual plants and inoculate them into blank MS medium for transition culture; when the test-tube seedlings are about 1.5-2.0 cm tall and have 15-20 thick leaves, they are acclimatized and transplanted.
[0014] According to a preferred embodiment, the embryogenic callus proliferation medium in step S1 is more preferably MS + 1.5 mg / L-2.0 mg / L 6-BA + 1.0 mg / L 2,4-D.
[0015] According to a preferred embodiment, the embryogenic callus proliferation medium in step S1 is more preferably MS + 1.5 mg / L 6-BA + 1.0 mg / L 2,4-D.
[0016] According to a preferred embodiment, in step S2, the callus-somatic embryo mixture proliferation medium is: MS + 1.0 mg / L-1.5 mg / L 6-BA + 0.5 mg / L-1.5 mg / L NAA + 0.5 mg / L-1.0 mg / L 2,4-D;
[0017] According to a preferred embodiment, the callus-somatic embryo mixture proliferation medium in step S2 is MS + 1.5 mg / L 6-BA + 0.5 mg / L NAA + 1.0 mg / L 2,4-D.
[0018] According to a preferred embodiment, the method further includes step S4, in which the flattened spherical clustered buds that have grown after seedling culture in step S3 are divided into individual plants and then inoculated into blank MS medium for transition culture. Between steps S2 and S3, step S2.1 is also included: the callus-somatic embryo mixture obtained in step S2 is divided into 2-3 seedlings with a small amount of embryogenic callus tissue and re-inoculated into the medium of step S2 for seedling culture; preferably, the small amount of embryogenic callus tissue is visible to the naked eye.
[0019] According to a preferred embodiment, the size of the "suitable inoculation piece" in step S2 is 0.5 cm-1.2 cm × 0.5 cm-1.2 cm.
[0020] According to a preferred embodiment, in the tissue culture of steps S1-S3, the temperature of the culture room is controlled at 22 ± 1℃, the light intensity is 1500-2000 lx, and the light exposure time is 10 h / d.
[0021] According to a preferred embodiment, before step S1, the method further includes the following step S0: selecting well-developed and robust leaves as explants, inoculating them into a sterile seedling culture medium for cultivation and expansion to obtain the first batch of sterile seedlings, and obtaining more explant material from the sterile seedlings. Preferably, the sterile seedling culture medium can be a commercially available general-purpose succulent plant culture medium that can achieve propagation.
[0022] According to a preferred embodiment, in step S0, the sterile seedling culture medium is: MS + 1.0 mg / L 6-BA + 1.0 mg / L NAA.
[0023] Compared with existing technologies, the advantages of this invention are:
[0024] 1. A method for germplasm preservation and commercial rapid propagation of Echeveria elegans. This application induces somatic embryos based on a combination of plant growth hormones without using TDZ, and the induction rate is high, with a proliferation coefficient of up to 7.5. At the same time, it avoids the problems of high cost, large proportion of deformed seedlings and low reproducibility associated with TDZ. No deformities or developmental delays were observed.
[0025] 2. A method for the preservation and rapid commercial propagation of *Echeveria elegans* germplasm. This application is based on somatic embryonic development, inducing somatic cells into somatic embryonic callus tissue, which significantly improves the propagation coefficient. The propagation coefficient reaches 6.5-7.5 during callus induction, and further increases to 9.5-12 after further proliferation culture. After 60 days of seedling culture, the seedling formation coefficient reaches 12.5. If leaves are used as material for further grafting at this point, the proliferation coefficient reaches 62.5; adding the 11.6 from the callus-somatic embryo mixture, the total proliferation coefficient reaches a considerable 70 or more.
[0026] 3. A method for germplasm preservation and commercial rapid propagation of Echeveria elegans. In this application, a highly efficient proliferation method using a mixture of callus and somatic embryo is adopted. In step S4, the seedlings that grow further after division culture are further separated into individual plants for seedling culture by physical separation, thereby increasing the seedling success rate from 3.1 without physical separation to 12.1.
[0027] 4. A method for germplasm preservation and rapid commercial propagation of *Echeveria elegans*. After seedling cultivation, the survival rate of seedlings after acclimatization is 100%, and the plants complete morphological development and maintain normal physiological states. Although the initial establishment of the system is relatively time-consuming, once established, further subculturing of the callus-somatic embryo mixture allows for induction from the callus-somatic embryo mixture without starting from explants. This method is simple to operate, has a high propagation coefficient, high seedling survival rate, and high efficiency, demonstrating significant advantages. This system not only provides technical support for germplasm preservation of *Echeveria elegans* but also provides theoretical reference for hormone ratio optimization and morphological regulation in the commercial propagation of succulent plants. Attached Figure Description
[0028] Figure 1 This diagram illustrates the process of establishing a sterile system. Figure A shows the growth after 30 days of culture; B shows the growth after 45 days of culture; C shows the growth after 60 days of culture; and D shows the growth after 75 days of culture. Scale bar = 0.5 cm.
[0029] Figure 2 The results of the embryogenic callus induction and proliferation experiment in step S2 are shown in the figure. A represents the growth of the CK experimental group after 60 days of culture; B represents the growth of the A5 experimental group after 60 days of culture; C represents the growth of the B5 experimental group after 60 days of culture; scale bar = 2.0 cm.
[0030] Figure 3This represents the proliferation process of embryogenic callus B5 in step S2; Figure A shows the growth after 15 days of culture; B shows the growth after 30 days of culture; C shows the growth after 45 days of culture; D shows the growth after 60 days of culture; scale bar = 2.0 cm;
[0031] Figure 4 This diagram shows the growth process of embryogenic callus in the optimal culture medium; Figure A shows the growth after 15 days of culture; B shows the growth after 30 days of culture; C shows the growth after 45 days of culture; D shows the growth after 60 days of culture; Scale bar = 2.0 cm;
[0032] Figure 5 This is a diagram showing the seedling growth process; A. Growth after 15 days of cultivation; B. Growth after 30 days of cultivation; C. Growth after 45 days of cultivation; D. Growth after 60 days of cultivation; Scale bar = 0.8 cm;
[0033] Figure 6 This diagram shows the transitional culture process of test-tube seedlings. Figure A shows the growth after 10 days of culture; B shows the growth after 20 days of culture; and C shows the growth after 30 days of culture. Scale bar = 1.8 cm.
[0034] Figure 7 For the results of transplanting and acclimatization of test-tube seedlings, A. Growth of test-tube seedlings after 30 days of cultivation in a foam box; B. Growth of test-tube seedlings after 60 days of transplanting to flower pots; Scale bar A = 4 cm; Scale bar B = 6 cm. Detailed Implementation
[0035] The specific embodiments listed in this invention are merely examples, and the invention is not limited to the specific embodiments described below. For those skilled in the art, any equivalent modifications and substitutions to the embodiments described below are also within the scope of this invention. Therefore, all equivalent transformations and modifications made without departing from the spirit and scope of this invention should be covered within its scope. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer shall apply. All reagents or instruments whose manufacturers are not specified are commercially available conventional products. To better illustrate this invention, numerous specific details are provided in the following detailed embodiments. Those skilled in the art should understand that this invention can be practiced even without certain specific details. In other embodiments, methods, means, equipment, and steps well known to those skilled in the art are not described in detail in order to highlight the main points of this invention.
[0036] Unless otherwise defined, all technical and scientific terms used herein have the same meanings as commonly understood by one of ordinary skill in the art. Unless otherwise specified, all units used in this specification are International Standard Units (SI), and all numerical values and ranges appearing in this invention should be understood to include systematic errors unavoidable in industrial production.
[0037] The features and performance of the present invention will be further described in detail below with reference to embodiments. Example 1
[0038] 1. Materials and Culture Media
[0039] 1.1 Materials
[0040] Five pots containing ten green-flowered stone lotus plants were provided by Yunnan Suifeng Jiayuan Agricultural Technology Co., Ltd., and the plants were identified by Yunnan University as... Sinocrassula indica var. viridiflora KT Fu. The sterilization reagent, mercuric chloride (HgCl2), was purchased from the Guizhou Tongren Chemical Research Institute, China. Other chemical reagents, including sodium hydroxide (NaOH), sucrose, agar, and plant growth regulators such as 2,4-dichlorophenoxyacetic acid (2,4-D), kinetin (KT), 1-naphthylacetic acid (NAA), indole-3-butyric acid (IBA), and 6-benzylpurine [N-(Phenylmethyl)-9H-purin-6-amine, 6-BA], were all analytical grade and purchased from Beijing Dingguo Biotechnology Co., Ltd. (Beijing, China). Unless otherwise specified in the study, all values are mass concentrations.
[0041] 1.2 Culture medium
[0042] The basic culture medium was MS, with 3.0% sucrose and 0.47% agar added. The pH was adjusted to 5.6-5.8 using 1 N NaOH solution. The culture was then sterilized in an autoclave at 123℃ for 25 min before use.
[0043] 1.3 Cultivation Conditions
[0044] The temperature in the culture room was controlled at 22 ± 1℃, the light intensity was 1500-2000 lx, and the light duration was 10 h / d.
[0045] 1.4 Data Processing
[0046] Measurement and analysis of various indicators
[0047] All data were processed and analyzed using Microsoft Office Excel (2019) and SPSS (29.0) software; images were created using Adobe Photoshop (2021) software.
[0048] Proliferation coefficient = Effective transfer number / Original inoculation number;
[0049] Seedling survival rate = Total number of seedlings / Total number of original inoculated individuals;
[0050] Survival rate (%) = Number of surviving plants / Total number of transplanted plants × 100%.
[0051] 2 Methods
[0052] A method for germplasm preservation and rapid commercial propagation of Echeveria 'Green Flower' includes the following steps:
[0053] Step S0: Aseptic seedling acquisition: Select healthy and robust leaves as explants, inoculate them into aseptic seedling culture medium to grow and expand the first batch of aseptic seedlings, and obtain more explant materials from the aseptic seedlings;
[0054] Specifically:
[0055] Healthy, well-developed leaves were selected as explants. After rinsing under running water for 30 minutes, they were sterilized by immersion in 0.1% mercuric chloride solution for 8 minutes in a clean bench, followed by rinsing three times with sterile water for at least 3 minutes each time. The leaves were then dried and inoculated into sterile seedling medium: MS + 1.0 mg / L 6-BA + 1.0 mg / L NAA. One material was used per bottle, and 30 bottles were inoculated. After obtaining the first batch of sterile seedlings, they were subcultured multiple times on the same fresh medium to increase the number of cultures.
[0056] The cultivation process is as follows Figure 1 As shown. After disinfection, the leaves were inoculated onto the starter culture medium. After 30 days of culture, the base of the leaves swelled to form a dense, green callus tissue with distinct bud points. Figure 1 A); After 45 days, one of the buds grew rapidly, with large and thick leaves that basically covered the entire culture, and white adventitious roots appeared at the same time. Figure 1 B); After 60 days, the main bud grows further, and the small buds suppressed at the base begin to differentiate ( Figure 1 C); After 75 days, the main bud stopped growing, and distinctive red spots appeared at the leaf tips. The basal buds also continued to grow, but this growth was significantly inhibited, and the adventitious roots also elongated significantly. Figure 1 D).
[0057] Step S1: Embryogenic callus proliferation: Explant material was obtained from the sterile seedlings obtained in step S1 and inoculated into embryogenic callus proliferation medium to further induce embryogenic callus.
[0058] Specifically:
[0059] Mature leaves from sterile seedlings in step S0 were used as explants and inoculated into embryogenic callus proliferation medium to induce embryogenic callus. Two complete combination experiments were conducted: 6-BA / NAA and 6-BA / 2,4-D. The concentrations of 6-BA were 1.0, 1.5, and 2.0 mg / L; the concentrations of NAA were 0.5, 1.0, and 1.5 mg / L; and the concentrations of 2,4-D were 0.5, 1.0, and 1.5 mg / L. Five bottles were inoculated per group, with 10 materials per bottle, and the experiments were repeated three times. After 60 days, the proliferation of embryogenic callus in each experimental group was observed, and the proliferation coefficient was calculated. If contamination occurred, the remaining material was replenished promptly, bottle by bottle. The effects of different hormone combinations on embryogenic callus proliferation are shown in Table 2 below.
[0060] Table 1. Effects of different hormone combinations on the proliferation of embryonic callus.
[0061]
[0062] Note: Different letters in the same column in the table indicate significant differences at the 0.05 level, and the same applies below.
[0063] In the control group (CK) without plant growth regulators, although the leaf base could swell to form a few buds and develop into small plantlets with adventitious roots, embryogenic callus formation was not induced. Figure 2 A). In all other combinations, embryogenic callus was successfully induced from the leaves, but the proliferation coefficient of embryogenic callus varied. Experimental data (Table 1) showed that when the 6-BA concentration was fixed, the proliferation coefficient of embryogenic callus first increased and then decreased with increasing NAA or 2,4-D concentrations, indicating that excessively high or low NAA and 2,4-D concentrations inhibited callus proliferation. When NAA or 2,4-D was in a low concentration range, the proliferation coefficient significantly increased with increasing 6-BA concentration, indicating that 6-BA promoted the proliferation of embryogenic callus. Comparing the two groups, the proliferation effect of the 6-BA / NAA combination was generally inferior to that of the 6-BA / 2,4-D combination. The optimal treatment group (A5 group, 1.5 mg / L 6-BA + 1.0 mg / L NAA) had a proliferation coefficient of 6.81, and the callus showed a yellow-green, dense structure with bud formation. Figure 2 B); the latter optimal treatment group (B5 group, 1.5 mg / L 6-BA + 1.0 mg / L 2,4-D) achieved a proliferation coefficient of 7.50, and the callus tissue was bright green, dense in texture, and had raised bud points on the surface. Figure 2 C).
[0064] In the B5 experimental group, after 15 days of culture, the leaf base swelled to produce loose, yellowish-green embryogenic callus ( Figure 3 A); After 30 days, embryogenic callus tissue significantly proliferated and its structure tended to become dense ( Figure 3 B); After 45 days, the embryogenic callus continued to proliferate, and green protrusions began to appear on the surface ( Figure 3 C); After 60 days, the callus tissue turned bright green and proliferated extensively, with a significant increase in the number of buds, but no complete plantlets were observed to form. Figure 3 D). The callus tissue of the leaves of *Sedum morganianum* is embryogenic callus tissue. Part of it continues to proliferate, while the other part transforms into embryonic cell clusters. In a suitable culture medium, a callus-somatic embryo mixture is formed and proliferates in large quantities. The somatic embryos develop into seedlings.
[0065] During the proliferation of embryogenic callus in *Gymnocalycium mihanovichii*, a white, frost-like substance appearing before somatic embryo differentiation exhibits stage-specific characteristics closely related to the developmental process. Observations revealed that the white frost-like substance is generated in the early stages of embryogenic callus formation, gradually accumulating during proliferation, and becoming most prominent at the peak of proliferation; it gradually disappears after the somatic embryo enters the organ differentiation stage. It is speculated that this substance may be a specific metabolite (such as polysaccharides or lipids) during embryogenic cell proliferation, and its functions may include: (1) forming a physical barrier to maintain embryogenic microenvironment homeostasis; (2) influencing intercellular signal transduction through isolation. The disappearance of the frost-like substance may be related to the remodeling of the extracellular matrix during the establishment of embryonic polarity, indicating that its metabolic clearance is an important triggering condition for somatic embryo differentiation. Similar phenomena are observed in… Coffea arabica L. var. Colombia In somatic embryogenesis systems, it has also been reported that white, compact callus tissue has a stronger somatic embryogenetic capacity compared to beige, soft callus tissue. However, Cucumis sativus This phenomenon was not observed in species such as L., suggesting that this characteristic is likely specific. Thus, the white, frosty substance could serve as a morphological indicator of the proliferative stage of embryogenic callus in *Lithocarpus spp.*
[0066] Step S2: Proliferation of callus-somatic embryo mixture: The callus-somatic embryo mixture obtained in step S1 was cut into 1.0 × 1.0 cm pieces and inoculated into the callus-somatic embryo mixture proliferation medium for proliferation.
[0067] Specifically:
[0068] The embryogenic callus obtained in step S1 was subjected to L9 (3) assay with 6-BA (A), NAA (B), and 2,4-D (C) as factors. 4Orthogonal experiments were conducted (Table 2). Five bottles were inoculated into each group, with 10 materials per bottle, and the experiments were repeated three times. After 60 days, the proliferation of callus-somatic embryo mixtures in each experimental group was observed, and the proliferation coefficient was calculated. If contamination occurred, the mixture was replenished promptly on a bottle-by-bottle basis.
[0069] Table 2. Proliferation of callus-somatic embryo mixture L9 (3) 4 Orthogonal experimental design
[0070]
[0071] The results are shown in Table 3 below:
[0072] Table 3. Proliferation of callus-somatic embryo mixture L9 (3) 4 Orthogonal experiment results
[0073]
[0074] Table 4. Results of ANOVA on the proliferation coefficient of callus-somatic embryo mixture.
[0075]
[0076] Table 5. Duncan's test at 6-BA level 3
[0077]
[0078] Orthogonal experiments revealed that the callus-somatic embryo mixture continued to grow, and the somatic embryos developed into seedlings, significantly increasing the proliferation coefficient of *Echeveria elegans*. The results of the orthogonal experiment analysis are shown in Tables 3, 4, and 5. Table 3 shows that... R A (1.51) > R C (0.59) > R B (0.45) > R D (0.33), 6-BA, NAA and 2,4-D R The values were all greater than the blank column, indicating that all three factors had a reliable effect on the proliferation of callus-somatic embryo mixtures, with 6-BA being the most significant influencing factor. The results of the ANOVA in Table 4 show that 6-BA had a statistically significant effect on the proliferation of callus-somatic embryo mixtures. P < 0.05), while NAA and 2,4-D showed no statistical significance ( P >0.05). Further analysis using a three-level Duncan test on 6-BA (Table 5) showed that level 2 (1.5 mg / L) was significantly more effective than level 1 (1.0 mg / L) and level 3 (2.0 mg / L), with level 2 (1.5 mg / L) being the most suitable. Analysis of the mean values indicated that the optimal hormone combination for inducing proliferation of *Echeveria elegans* callus-somatic embryo mixture was A2B1C2 (1.5 mg / L 6-BA + 0.5 mg / L NAA + 1.0 mg / L 2,4-D).
[0079] After culturing in the above-mentioned optimal culture medium for 15 days, the callus-somatic embryo mixture began to proliferate, and green protrusions appeared on the surface. Figure 4 A); After 30 days, the callus-somatic embryo mixture continued to proliferate, the number and size of the protrusions increased, and white adventitious roots appeared ( Figure 4 B); After 45 days, the volume of the mixture increased significantly, a few protrusions grew rapidly and developed into complete plants, and the number of roots increased ( Figure 4 C); After 60 days, the entire surface of the culture medium was covered by the callus-somatic embryo mixture, and the roots also covered the entire bottom of the bottle. At this time, the proliferation coefficient was 11.6 ( Figure 4 D). Although the callus-somatic embryo mixture can achieve good proliferation, the seedling coefficient calculated for the optimal culture medium is low, at only 3.1.
[0080] Step S3: Divide the callus-somatic embryo mixture obtained in step S2 into 2-3 seedlings and reintroduce them with a small amount of embryogenic callus tissue into the callus-somatic embryo mixture of step S2 for seedling culture; the remaining mixture is inoculated into fresh original culture medium in the same way for re-proliferation.
[0081] During the seedling culture stage, after 15 days of culture, the somatic embryos on the surface of the residual callus tissue rapidly differentiated into buds at different developmental stages. Figure 5 A); at 30 days, the bud population continued to expand, and the newly differentiated somatic embryos further developed into buds, with some plants showing red spots at the leaf tips ( Figure 5 B); After 45 days, the bud cluster morphology transformed into a flattened spherical structure, with the surface covered by numerous intact small plantlets, accompanied by the formation of white filamentous adventitious roots (B). Figure 5 C); After 60 days, the bud clusters become nearly spherical due to continuous growth, the plants develop normally, the leaves form 2-3 layers of thick structure and the leaf tips become deeper red, but root development is relatively slow, and the seedling survival rate reaches 12.5 ( Figure 5D). At this point, each complete plant has an average of 5 leaves, resulting in a total of 625 leaves. If the leaves are used as material for further grafting, the proliferation coefficient is 62.5. Adding the 11.6 from the callus-somatic embryo mixture, the proliferation coefficient reaches a considerable 70 or more.
[0082] Step S4: The flattened spherical clustered buds in the seedling culture were divided into individual plants and inoculated into blank MS medium for transition culture; after 10 days of culture, the volume of the test-tube seedlings increased, and the white root tips increased significantly. Figure 6 A); After 20 days, the test-tube seedlings continued to grow, the red spots on the leaf tips deepened in color, and the number and length of adventitious roots increased significantly. Figure 6 B); After 30 days, the test-tube seedlings grew vigorously, with well-developed root systems, and the red spots on the leaf tips tended to stabilize in color. Figure 6 C).
[0083] When the test-tube seedlings reach a height of approximately 1.5-2.0 cm and have 15-20 thick leaves, they are ready for acclimatization and transplanting. The culture bottles are first placed indoors for 5 days to harden off, then moved outdoors for 10 days. Afterward, the culture bottles are opened, and the seedlings are removed and their roots are washed with tap water to remove the culture medium. They are then soaked in a 0.1% carbendazim solution for 5 minutes. The seedlings are then transplanted into foam boxes (inner dimensions: 13×18×15cm) containing a 3-5 cm thick layer of soil substrate (humus: perlite = 3:1 v / v) that has been sterilized in an autoclave at 125℃ for 30 minutes. The substrate is thoroughly watered and sealed with plastic wrap to maintain warmth and humidity (temperature 25±2℃, relative humidity 60%-80%). The sealed foam boxes are then placed in a greenhouse for cultivation. After 30 days, the seedling survival rate is assessed, and the healthy seedlings are transplanted into round flowerpots with a diameter of 18.5 cm.
[0084] During the acclimatization and cultivation stage, the survival rate of the test-tube seedlings was 100% after 30 days of culture in foam boxes, and the plants completed morphogenesis and maintained normal physiological state. Figure 7 A); 60 days after transplanting into a round pot, the plant grew vigorously, and the leaves showed a typical rosette arrangement, exhibiting stable ornamental characteristics. Figure 7 B).
[0085] The embodiments described above merely illustrate specific implementation methods of this application, and while the descriptions are detailed and specific, they should not be construed as limiting the scope of protection of this application. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the technical solution of this application, and these modifications and improvements all fall within the scope of protection of this application.
Claims
1. A method for germplasm preservation and rapid commercial propagation of *Echeveria elegans*, characterized in that, Includes the following steps: Step S1: Embryogenic callus proliferation: Mature leaves were obtained from sterile seedlings as explant material and inoculated into embryogenic callus proliferation medium to induce embryogenic callus. The culture medium for the proliferation of embryogenic callus is: MS + 1.0 mg / L-2.0 mg / L 6-BA + 0.5 mg / L-1.0 mg / L NAA or MS + 1.0 mg / L-2.0 mg / L 6-BA + 0.5 mg / L-1.0 mg / L 2,4-D; Step S2: Proliferation of callus-somatic embryo mixture: The embryogenic callus tissue obtained in step S1 is further cultured to form a callus-somatic embryo mixture. The obtained callus-somatic embryo mixture is cut into small pieces suitable for inoculation and inoculated into a callus-somatic embryo mixture proliferation medium for proliferation induction to obtain cluster shoots. The callus-somatic embryo mixture proliferation medium is: MS + 1.0 mg / L-1.5 mg / L 6-BA + 0.5 mg / L-1.5 mg / L NAA + 0.5 mg / L-1.0 mg / L 2,4-D. Step S3: Divide the clustered shoots induced in step S2 into individual plants and inoculate them into blank MS medium for transition culture; when the test-tube seedlings are 1.5-2.0 cm tall and have 15-20 thick leaves, they are acclimatized and transplanted.
2. The method for germplasm preservation and rapid commercial propagation of *Echeveria elegans* according to claim 1, characterized in that, In step S1, the culture medium for the proliferation of embryogenic callus is MS + 1.5 mg / L-2.0 mg / L 6-BA + 1.0 mg / L 2,4-D.
3. The method for germplasm preservation and rapid commercial propagation of *Echeveria elegans* according to claim 2, characterized in that, In step S1, the culture medium for the proliferation of embryogenic callus is MS + 1.5 mg / L 6-BA + 1.0 mg / L 2,4-D.
4. The method for germplasm preservation and rapid commercial propagation of *Echeveria elegans* according to claim 3, characterized in that, In step S2, the callus-somatic embryo mixture proliferation medium was MS + 1.5 mg / L 6-BA + 0.5 mg / L NAA + 1.0 mg / L 2,4-D.
5. The method for germplasm preservation and rapid commercial propagation of *Echeveria elegans* according to claim 1, characterized in that, Between steps S2 and S3, there is also step S2.1: the callus-somatic embryo mixture obtained in step S2 is divided into 2-3 seedlings and re-inoculated with a small amount of embryogenic callus tissue into the callus-somatic embryo mixture proliferation medium in step S2 for seedling culture.
6. The method for germplasm preservation and rapid commercial propagation of *Echeveria elegans* according to claim 1, characterized in that, The size of the "suitable inoculation piece" in step S2 is 0.5 cm-1.2 cm × 0.5 cm-1.2 cm.
7. The method for germplasm preservation and rapid commercial propagation of *Echeveria elegans* according to claim 1, characterized in that, In the tissue culture steps S1-S3, the temperature of the culture room was controlled at 22 ± 1 ℃, the light intensity was 1500-2000 lx, and the light duration was 10 h / d.
8. The method for germplasm preservation and rapid commercial propagation of *Echeveria elegans* according to claim 1, characterized in that, Before step S1, the following step S0 is also included: selecting well-grown and robust leaves as explants, inoculating them into a sterile seedling culture medium to grow and expand the first batch of sterile seedlings, and obtaining more explant materials from the sterile seedlings.
9. The method for germplasm preservation and rapid commercial propagation of *Echeveria elegans* according to claim 8, characterized in that, In step S0, the sterile seedling culture medium is: MS + 1.0 mg / L 6-BA + 1.0 mg / L NAA.