Germplasm preservation and commercialized rapid propagation method for Lepidogrammitis virens

Abstract

The invention relates to the technical field of plant tissue culture, and discloses a germplasm preservation and commercialized rapid propagation method for Lepidogrammitis virens, which comprises the following steps: step S1, inducing by taking mature leaves as explants to obtain embryonic calluses; the proliferation coefficient of the induced embryonic callus is up to 7.50; s2, cutting the callus-somatic embryo mixture obtained in the step S1, inoculating the cut callus-somatic embryo mixture into a callus-somatic embryo mixture proliferation culture medium, and carrying out proliferation to obtain cluster buds, wherein the proliferation coefficient reaches 11.65; s3, dividing the cluster buds into single plants, and inoculating the single plants into a blank MS culture medium for transition culture; and the seedling coefficient is increased from 3.1 to 12.5 by separating the cluster buds and independently culturing the cluster buds. S4, domesticating and transplanting. By the adoption of the rapid propagation method, the total propagation coefficient can reach 70.0 or above, the survival rate after domestication and transplanting reaches 100%, and the stable ornamental value is achieved.

Description

Technical Field

[0001] The present invention relates to the technical field of plant tissue culture and propagation, and in particular to a method for preserving and commercializing rapid propagation of Echeveria oleifera germplasm. Background Art

[0002] The statements in this section merely provide background information related to the present disclosure and may not constitute prior art.

[0003] Green flower stone lotus ( Sinocrassula indica var. viridiflora KT Fu) is a member of the Crassulaceae family (Echeveria genus). Sinocrassula Green Echeveria is a perennial herbaceous plant found primarily in limestone regions of central and northern Yunnan at altitudes of 1,500-3,000 meters, including Dali, Lijiang, and Kunming. Green Echeveria grows primarily in rock crevices or on steep cliffs, with patchy distributions and a unique and fragile habitat. According to recent surveys, Green Echeveria is scarce and vulnerable to invasive alien species, with its wild population classified as Vulnerable (VU). Green Echeveria has important ecological and ornamental value. Its unique rosette-shaped plant and gray-green leaves are highly ornamental, making it a valuable material for alpine succulent landscaping. Its leaves are rich in flavonoids, which exhibit significant antioxidant activity, and its potential medicinal properties warrant further exploration. Furthermore, as a pioneer plant in limestone regions, Green Echeveria plays a vital role in desertification control. Its roots secrete organic acids to dissolve rock and promote soil formation.

[0004] Succulent plants have two modes of reproduction in nature: sexual and asexual. Although sexual reproduction can produce variation, which is beneficial to evolution and adaptation, its seed germination rate is low and the cycle is long, and it takes 2-3 years or even longer to bloom; while vegetative reproduction such as bulbils, runners, and leaves can maintain the characteristics of the mother plant, but the reproduction coefficient is limited and it is extremely susceptible to diseases and pests. The artificial propagation methods of succulent plants mainly include cuttings, grafting, and tissue culture. Cutting propagation is simple to operate and has a survival rate of about 90%, but it is restricted by season and has a limited reproduction coefficient; grafting can quickly obtain excellent traits, but it has high technical requirements and the interface is prone to infection. Tissue culture can achieve large-scale rapid reproduction, with an annual reproduction coefficient of up to 10 5 -10 6 , demonstrating its broad prospects for commercial application in succulents. In recent years, significant breakthroughs have been made in in vitro culturing of succulents, primarily focusing on explant selection, regeneration system optimization, and culture environment regulation. While some progress has been made, most studies still rely on TDZ-induced somatic embryo formation, leading to problems such as high costs, a high 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, using explants such as leaves to directly differentiate into adventitious buds without going through the callus stage; (2) indirect organogenesis, explants such as leaves and inflorescences first form callus tissue, and then the callus tissue is induced to differentiate into adventitious buds and roots through plant growth regulators; (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; indirect somatic embryogenesis - somatic cells first dedifferentiate into callus tissue, and then some cells in the callus tissue differentiate into embryos. Among these three pathways, the direct organogenesis pathway does not require the callus tissue stage and directly differentiates buds or roots from explants, which can shorten the culture cycle and reduce the probability of contamination in long-term callus culture. However, it is only suitable for succulents with strong regeneration ability and is less efficient for species that are difficult to regenerate. However, it requires the use of young and healthy explants; the regeneration ability of old leaves or damaged tissues is significantly reduced. Indirect organogenesis has wide applicability. Through the callus stage, it can overcome the limitations of direct regeneration from explants, making it suitable for succulents that have difficulty regenerating. Callus tissue can be preserved and proliferated over long periods of time, making it suitable for commercial production. However, it also suffers from the problem of long culture cycles. Somatic embryogenesis has high regeneration efficiency, but requires strict regulation of hormones and culture conditions, otherwise it is prone to problems such as malformed embryos or developmental arrest.

[0006] The prior art document with publication number CN106613988B discloses a method for rapidly cultivating commercial small potted plants of the genus Echeveria that are difficult to propagate by cuttings by shaping in a bottle, wherein leaf tissue containing axillary bud meristems at the leaf base is used as a sterile induction explant, and method (1) direct organogenesis pathway is adopted, wherein an 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 5.5-6.0) is used to directly induce cluster buds without going through the callus tissue stage; the induced cluster buds are cultured using a proliferation medium (MS+ZT 0.2-0.3 mg / L+NAA 0.1-0 .2mg / L + 30-50mg / L adenine sulfate + 25-30g / L sucrose + 300-500mg / L activated carbon + 6.0-7.0g / L agar, pH 5.5-6.0) for induction of proliferation; subsequently, rooting induction is performed using seedling rooting medium (1 / 2MS + 0.3-0.5mg / L NAA + 25-30g / L sucrose + 300-500mg / L activated carbon + 6.0-7.0g / 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. This method bypasses the callus stage, resulting in a low propagation coefficient and a limited number of seedlings obtained within a given period. Summary of the Invention

[0007] The present invention aims to address the problems existing in the existing technologies and provide a method for the preservation and commercial rapid propagation of Echeveria glabra. By establishing a sterile culture system using mature leaves as explants, the effects of the embryonic callus induction medium, somatic embryogenesis, and plant regeneration medium of Echeveria glabra are optimized, successfully inducing embryonic callus and achieving efficient proliferation, thereby establishing a stable and efficient regeneration system. The research results have both urgent and ecological and economic value for the conservation of the germplasm resources and the sustainable commercial utilization of Echeveria glabra, a high-value endemic plant.

[0008] The technical solutions of the present invention are as follows: A method for preserving and commercializing the rapid propagation of green flower echeveria germplasm comprises the following steps: Step S1: embryonic callus proliferation: obtaining explant material from sterile seedlings and inoculating it into embryonic callus proliferation medium to induce embryonic callus; The embryonic callus proliferation medium is: 1.0 mg / L-2.0 mg / L 6-BA + 0.5 mg / L-1.0 mg / L NAA or 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 the callus-somatic embryo mixture: The embryonic callus obtained in step S1 further develops into a callus-somatic embryo mixture. The callus-somatic embryo mixture obtained in step S1 is cut into small pieces suitable for inoculation, and then inoculated into a callus-somatic embryo mixture proliferation medium for proliferation induction to obtain clustered shoots. Step S3: The clustered buds induced in step S2 are divided into individual plants and then inoculated into a blank MS medium for transitional culture; when the test tube seedlings are about 1.5-2.0 cm tall and have 15-20 thick leaves, they are acclimated and transplanted.

[0009] According to a preferred embodiment, the embryonic callus proliferation medium in step S1 is more preferably 1.5 mg / L-2.0 mg / L 6-BA + 1.0 mg / L 2,4-D.

[0010] According to a preferred embodiment, the embryonic callus proliferation medium in step S1 is more preferably 1.5 mg / L 6-BA + 1.0 mg / L 2,4-D.

[0011] According to a preferred embodiment, in step S2, the callus-somatic embryo mixture proliferation medium is: 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; According to a preferred embodiment, the callus-somatic embryo mixture proliferation culture medium in step S2 is 1.5 mg / L 6-BA + 0.5 mg / L NAA + 1.0 mg / L 2,4-D.

[0012] According to a preferred embodiment, in step S4, the oblate spherical clustered buds grown after the 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, there is also step S2.1: dividing the callus-somatic embryo mixture obtained by proliferation in step S2 into 2-3 seedlings and re-inoculating them with a small amount of embryonic callus tissue in the medium of step S2 for seedling culture; preferably, the small amount of embryonic callus tissue is visible to the naked eye.

[0013] According to a preferred embodiment, the "small piece suitable for inoculation" in step S2 is 0.5 cm-1.2 cm×0.5 cm-1.2 cm.

[0014] According to a preferred embodiment, during the tissue culture in steps S1-S3, the temperature of the culture chamber is controlled at 22±1°C, the light intensity is 1500-2000 lx, and the illumination time is 10 h / d.

[0015] According to a preferred embodiment, the method further includes the following step S0 before step S1: selecting well-developed, robust leaves as explants, inoculating them into a sterile seedling culture medium, and culturing and expanding them to obtain a first batch of sterile seedlings, and obtaining more explant material from the sterile seedlings. Preferably, the sterile seedling culture medium can be a general succulent plant culture medium currently available on the market that can achieve propagation.

[0016] 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.

[0017] Compared with the existing technology, the present invention has the following beneficial effects: 1. A method for the preservation and commercial rapid propagation of green flower Echeveria germplasm. This invention, without the use of TDZ, induces somatic embryos based on a combination of plant growth hormones, with a high induction rate and a proliferation coefficient of up to 7.5. It also avoids the high cost, high proportion of deformed seedlings, and low reproducibility of TDZ, and does not cause deformities or developmental delays. 2. A method for the preservation and commercial rapid propagation of green flower stone lotus germplasm. This application is based on the somatic embryo development method. The somatic cells are first induced to become somatic embryonic callus, which significantly improves the reproduction coefficient. During the callus induction process, the reproduction coefficient reaches 6.5-7.5. After further proliferation culture, the callus reproduction coefficient further reaches 9.5-12. After 60 days of seedling culture, the seedling coefficient reaches 12.5. If leaves are used as the material for further transfer at this time, the reproduction coefficient is 62.5. When combined with the 11.6 of the callus-somatic embryo mixture, the reproduction coefficient reaches a considerable 70 or above.

[0018] 3. A method for the preservation and commercial rapid propagation of green flower stone lotus germplasm. In this application, the efficient proliferation of the callus-somatic embryo mixture is adopted, and the seedlings grown after the division culture in step S4 are further divided into single plants by physical separation for seedling culture, thereby increasing the seedling coefficient from 3.1 without physical separation to 12.1.

[0019] 4. A method for the preservation and commercial rapid propagation of green Echeveria germplasm. After acclimation and cultivation, the survival rate of seedlings after seedling cultivation is 100%, and the plants complete morphological development and maintain normal physiological state. Although the initial establishment of the system is relatively time-consuming, once the system is established, through further subculture and proliferation of the callus-somatic embryo mixture, subsequent culture processes can achieve further induction from the callus-somatic embryo mixture without starting from explants. This system is simple to operate, has a high propagation coefficient and high seedling rate, and takes efficiency into consideration, with significant advantages. This system not only provides technical support for the preservation of green Echeveria germplasm, but also provides a theoretical reference for the optimization of hormone ratios and morphological regulation in the commercial propagation of succulents. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 The process of establishing a sterile system. Figure A shows growth after 30 days of culture; B shows growth after 45 days of culture; C shows growth after 60 days of culture; D shows growth after 75 days of culture. Scale bar = 0.5 cm. Figure 2 Results of the embryonic callus induction and proliferation test in step S2; Figure A shows the growth of the CK test group after 60 days of culture; Figure B shows the growth of the A5 test group after 60 days of culture; Figure C shows the growth of the B5 test group after 60 days of culture; Scale bar = 2.0 cm; Figure 3 This figure shows the proliferation process of embryonic callus tissue in group B5 in step S2. A. Growth after 15 days of culture; B. Growth after 30 days of culture; C. Growth after 45 days of culture; D. Growth after 60 days of culture; scale bar = 2.0 cm. Figure 4 The growth process of embryonic callus in the optimal culture medium; A. Growth after 15 days of culture; B. Growth after 30 days of culture; C. Growth after 45 days of culture; D. Growth after 60 days of culture; Scale bar = 2.0 cm; Figure 5 Diagram showing the seedling stage; A. Growth after 15 days of culture; B. Growth after 30 days of culture; C. Growth after 45 days of culture; D. Growth after 60 days of culture; scale bar = 0.8 cm; Figure 6 Figure 1 shows the transitional culture process of test tube seedlings. A. Growth after 10 days of culture; B. Growth after 20 days of culture; C. Growth after 30 days of culture. Scale bar = 1.8 cm. Figure 7Results of test-tube seedling acclimation and transplanting: A. Growth of test-tube seedlings after 30 days of culture in a foam box; B. Growth of test-tube seedlings after 60 days of transplanting into pots; Scale A = 4 cm; Scale B = 6 cm. DETAILED DESCRIPTION

[0021] The specific embodiments listed in the present invention are only examples of the present invention, and the present 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 the present invention. Therefore, equivalent transformations and modifications made without departing from the spirit and scope of the present invention should be included within the scope of the present invention. If specific conditions are not specified in the embodiments, they are carried out according to conventional conditions or conditions recommended by the manufacturer. All reagents or instruments without the manufacturer specified are conventional products that can be purchased commercially. In order to better illustrate the present invention, numerous specific details are given in the specific embodiments below. Those skilled in the art should understand that the present invention can also be implemented 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 purpose of the present invention.

[0022] Unless otherwise defined, all technical and scientific terms used herein have the same meanings as commonly understood by those skilled in the art. Unless otherwise specified, the units used in this specification are International Standard Units, and the numerical values ​​and numerical ranges appearing in this invention should be understood to include the inevitable systematic errors in industrial production.

[0023] The features and performance of the present invention are further described in detail below with reference to the embodiments. Example

[0024] 1 Materials and culture medium 1.1 Materials 5 pots of 10 green flower Echeveria plants were provided by Yunnan Suifeng Jiayuan Agricultural Technology Co., Ltd. The plants were identified by Yunnan University as Sinocrassula indica var. viridifloraThe sterilizing reagent mercuric chloride (HgCl2) was purchased from the Tongren Chemical Research Institute, Guizhou, China. The remaining chemical reagents, including sodium hydroxide (NaOH), sucrose, agar, and the plant growth regulators 2,4-dichlorophenoxyacetic acid (2,4-D), kinetin (KT), naphthaleneacetic acid (NAA), indole-3-butyric acid (IBA), and 6-benzylpurine [N-(Phenylmethyl)-9H-purin-6-amine, 6-BA], were all analytically pure and purchased from Beijing Dingguo Biotechnology Co., Ltd. (Beijing, China). Unless otherwise specified, all concentrations are expressed as mass.

[0025] 1.2 Culture medium The basic culture medium was MS, supplemented with 3.0% sucrose and 0.47% agar, and the pH was adjusted to 5.6-5.8 using 1 N NaOH solution. The culture medium was sterilized in a high-pressure steam sterilizer at 123°C for 25 min before use.

[0026] 1.3 Culture conditions The temperature of the culture room was controlled at 22 ± 1°C, the light intensity was 1500-2000 lx, and the photoperiod was 10 h / d.

[0027] 1.4 Data Processing Measurement and analysis of various indicators The data were processed and analyzed using Microsoft Office Excel (2019) and SPSS (29.0) software; the images were produced using Adobe Photoshop (2021) software.

[0028] Multiplication coefficient = effective transfer number / original inoculum number; Seedling coefficient = total number of seedlings / total number of original inoculations; Survival rate (%) = number of surviving plants / total number of transplanted plants × 100%.

[0029] 2 Methods A method for preserving and commercializing the rapid propagation of green flower echeveria germplasm comprises the following steps: Step S0: Obtaining sterile seedlings: selecting leaves with good growth and development and being robust as explants, inoculating them into sterile seedling culture medium, culturing and expanding them to obtain the first batch of sterile seedlings, and obtaining more explant materials from the sterile seedlings; Specifically: Select healthy, well-developed leaves as explants. Rinse them under running water for 30 minutes, then disinfect them by soaking them in 0.1% mercuric chloride solution in a clean bench for 8 minutes. Rinse them three times with sterile water, each time for at least 3 minutes. Drain dry and inoculate them onto sterile seedling medium: MS + 1.0 mg / L 6-BA + 1.0 mg / L NAA. Inoculate one material per bottle, and inoculate 30 bottles. After obtaining the first batch of sterile seedlings, transfer them to fresh medium and subculture multiple times to expand the number of cultures.

[0030] The cultivation process is as follows Figure 1 As shown. The leaves were sterilized and inoculated on the initiation medium. After 30 days of culture, the base of the leaves swelled to form a dense green callus with obvious buds ( Figure 1 A); After 45 days, a small bud in the bud point grew rapidly, its leaves were large and thick, basically covering the entire culture, and white adventitious roots appeared ( Figure 1 B); After 60 days, the main bud grows further and the small buds suppressed by it begin to differentiate ( Figure 1 C); After 75 days, the main bud stopped growing, and a distinctive red dot appeared on the tip of its leaf. The small buds at the base also grew further, but were obviously inhibited, and the adventitious roots also elongated significantly ( Figure 1 D).

[0031] Step S1: Embryogenic callus proliferation: Explant material is obtained from the sterile seedlings obtained in step S1 and inoculated into an embryonic callus proliferation medium to further induce embryonic callus.

[0032] Specifically: Take the mature leaves of the sterile seedlings in step S0 as explants and inoculate them into the embryonic callus proliferation medium to induce embryonic callus. Two complete combination tests of 6-BA / NAA and 6-BA / 2,4-D were carried out respectively, with 6-BA concentrations of 1.0, 1.5, and 2.0 mg / L, NAA concentrations of 0.5, 1.0, and 1.5 mg / L, and 2,4-D concentrations of 0.5, 1.0, and 1.5 mg / L. Each group was inoculated with 5 bottles, 10 materials per bottle, and repeated 3 times. After 60 days, the proliferation of embryonic callus in each test group was observed, and the proliferation coefficient was calculated. If contamination occurred, it was replenished in time per bottle. The effects of different hormone combinations on the proliferation of embryonic callus are shown in Table 2 below: Table 1 Effects of different hormone combinations on embryogenic callus proliferation

[0033] Note: Different letters in the same column in the table indicate significant differences at the 0.05 level, the same below.

[0034] In the CK group without the addition of plant growth regulators, although the base of the leaves could swell to form a small number of buds and develop into small plants with adventitious roots, embryonic callus formation could not be induced ( Figure 2 A). In all other combinations, embryonic callus was successfully induced from the leaves, but there were differences in the reproduction coefficients of the embryonic callus. Experimental data (Table 1) showed that when the 6-BA concentration was fixed, the embryonic callus proliferation coefficient showed a trend of first increasing and then decreasing as the NAA or 2,4-D concentration increased, indicating that too high or too 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 increased significantly as the 6-BA concentration increased, indicating that 6-BA promoted the proliferation of embryonic callus. Comparing the two groups of experiments, the proliferation effect of the 6-BA / NAA combination was overall inferior to that of 6-BA / 2,4-D. The proliferation coefficient of the former optimal treatment group (A5 group, 1.5 mg / L 6-BA + 1.0 mg / L NAA) was 6.81, and the callus showed a yellow-green compact structure with bud formation ( Figure 2 B); the latter optimal treatment group (B5, 1.5 mg / L 6-BA + 1.0 mg / L 2,4-D) had a proliferation coefficient of 7.50, and the callus was bright green, dense in texture, and had buds on the surface ( Figure 2 C).

[0035] In the B5 experimental group, after 15 days of culture, the base of the leaves swelled and produced loose, yellow-green embryonic callus ( Figure 3 A); After 30 days, the embryonic callus proliferated significantly and the structure became dense ( Figure 3 B); After 45 days, the embryonic 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 rapidly, and the number of buds increased significantly, but no complete plant was formed ( Figure 3 D). The callus tissue of the leaves of Echeveria oleifera is embryonic callus. Part of it continues to proliferate, while the other part transforms into embryonic cell clusters. In an appropriate culture medium, a callus-somatic embryo mixture is formed and proliferates in large quantities. The somatic embryos develop into seedlings.

[0036] During the proliferation of embryonic callus of Echeveria oleracea, the white frost that appears before the differentiation of somatic embryos shows stage characteristics that are closely related to the developmental process. It was observed that the white frost is generated in the early stage of embryonic callus formation, gradually accumulates with the proliferation process, and is most obvious at the peak of proliferation; when the somatic embryo enters the organ differentiation stage, the frost gradually disappears. It is speculated that this substance may be a specific metabolite (such as polysaccharides or lipid compounds) in the process of embryonic cell proliferation, and its functions may include: (1) forming a physical barrier to maintain the homeostasis of the embryonic microenvironment; (2) affecting intercellular signal transmission through isolation. The disappearance of the frost may be related to the reconstruction 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 have been reported in Coffea arabica L. var. Colombia It has also been reported in the somatic embryogenesis system of that white compact callus has stronger somatic embryogenesis ability than beige soft callus. Cucumis sativus L., suggesting that this characteristic is likely to be specific. Thus, the white frost can be used as a morphological indicator of the proliferation stage of embryonic callus in Echeveria glabra.

[0037] Step S2: Proliferation of callus-somatic embryo mixture: The callus-somatic embryo mixture obtained in step S1 was cut into pieces of 1.0 × 1.0 cm in size and inoculated into callus-somatic embryo mixture proliferation medium for proliferation.

[0038] Specifically: The embryogenic callus obtained in step S1 was subjected to L9 (3 4 ) orthogonal experiment (Table 2). Five bottles were inoculated per group, with 10 samples per bottle, and replicated three times. After 60 days, the proliferation of the callus-somatic embryo mixture 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.

[0039] Table 2 Proliferation of callus-somatic embryo mixture L9 (3 4 ) Orthogonal experimental design

[0040] The results are shown in Table 3 below: Table 3 Proliferation of callus-somatic embryo mixture L9 (3 4 ) Orthogonal test results

[0041] Table 4 Results of variance analysis of proliferation coefficient of callus-somatic embryo mixture

[0042] Table 5 6-BA 3-level Duncan test

[0043] The orthogonal test found that the callus-somatic embryo mixture continued to grow and the somatic embryos developed into seedlings, greatly improving the proliferation coefficient of green flower stone lotus. The results of the orthogonal test analysis are shown in Table 3, Table 4 and Table 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 ​​are all greater than the blank column, indicating that these three factors have a reliable effect on the proliferation of callus-somatic embryo mixture, and 6-BA is the most important factor affecting proliferation. The results of variance analysis in Table 4 show that 6-BA has a statistically significant effect on the proliferation of callus-somatic embryo mixture ( P < 0.05), NAA and 2,4-D had no statistical significance ( P > 0.05). Further three-level Duncan tests for 6-BA (Table 5) revealed that 6-BA level 2 (1.5 mg / L) was significantly more effective than levels 1 (1.0 mg / L) and 3 (2.0 mg / L), with level 2 (1.5 mg / L) being the most effective. Mean analysis revealed that the optimal hormone combination for inducing proliferation of Echeveria callus-somatic embryo mixtures was A2B1C2 (1.5 mg / L 6-BA + 0.5 mg / L NAA + 1.0 mg / L 2,4-D).

[0044] After culturing in the above optimal medium for 15 days, the callus-somatic embryo mixture began to proliferate, and green protrusions appeared on the surface ( Figure 4 A); 30 days later, the callus-somatic embryo mixture continued to proliferate, the number and volume of 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 effect, the seedling coefficient calculated in the optimal culture medium is low, only 3.1.

[0045] Step S3: the callus-somatic embryo mixture obtained by proliferation in step S2 is divided into 2-3 seedlings and a small amount of embryonic callus tissue is re-inoculated into the callus-somatic embryo mixture in step S2 for seedling induction culture; the remaining mixture is inoculated into fresh original culture medium in the same manner as before for re-proliferation; In the seedling culture stage, after 15 days of culture, the somatic embryos on the surface of the residual callus tissue rapidly differentiated into young shoots at different developmental stages ( Figure 5 A); At 30 days, the bud population continued to expand, the newly differentiated somatic embryos further developed into buds, and some plants showed red spots on the leaf tips ( Figure 5 B); After 45 days, the bud cluster morphology changed to a flat spherical structure, with a large number of complete plantlets on the surface, accompanied by the formation of white filamentous adventitious roots ( Figure 5 C); After 60 days, the bud cluster showed a nearly spherical shape due to continuous growth, the plant developed normally, the leaves formed 2-3 layers of thick structure and the red color of the leaf tips deepened, but the root system developed relatively slowly. At this time, the seedling coefficient reached 12.5 ( Figure 5 D). At this point, each complete plant has an average of 5 leaves, for a total of 625 leaves. If leaves are used for further grafting, the proliferation coefficient is 62.5. Adding the 11.6 of the callus-somatic embryo mixture, the proliferation coefficient reaches a respectable 70 or above.

[0046] Step S4: The oblate spherical clustered buds in the seedling culture were divided into individual plants and inoculated into blank MS medium for transitional culture. After 10 days of culture, the volume of the test tube seedlings increased and the number of 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, the number of adventitious roots increased, and the length was significantly extended ( Figure 6 B); After 30 days, the test tube seedlings grew robustly, with well-developed root systems, and the red spots on the leaf tips became stable in color ( Figure 6 C).

[0047] When the test-tube seedlings are approximately 1.5-2.0 cm tall and have 15-20 thick leaves, they are acclimated and transplanted. The culture bottles are first placed indoors for 5 days and then transferred outdoors for 10 days. The bottles are then opened, the test-tube seedlings are removed, the root medium is washed under tap water, and then soaked in a 0.1% carbendazim solution for 5 minutes. The seedlings are then transplanted into foam boxes (13 × 18 × 15 cm inner diameter) filled with a soil matrix (humus soil: perlite = 3:1, v / v) sterilized at 125°C for 30 minutes. The soil matrix is ​​covered with a 3-5 cm layer of plastic wrap, watered thoroughly, and sealed with plastic wrap to maintain heat and moisture retention (temperature 25 ± 2°C, relative humidity 60%-80%). The sealed foam boxes are then placed in a greenhouse for incubation. After 30 days, the survival rate of the seedlings is determined, and well-grown plants are transplanted into round pots with a diameter of 18.5 cm.

[0048] During the acclimation stage, the survival rate of the test tube seedlings after 30 days of foam box culture was 100%, and the plants completed morphological development and maintained normal physiological state ( Figure 7 A); 60 days after transplanting to the round pot, the plant grew robustly, with leaves arranged in a typical rosette pattern, showing stable ornamental characteristics ( Figure 7 B).

[0049] The above-described embodiments merely represent specific implementation methods of the present application. While the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of protection of the present application. It should be noted that a person skilled in the art would be able to make numerous variations and improvements without departing from the technical concept of the present application, and all such variations and improvements fall within the scope of protection of the present application.

Claims

1. A method for the preservation and commercial rapid propagation of Echeveria oleifera germplasm, characterized in that: The steps include: Step S1: embryonic callus proliferation: obtaining mature leaves from sterile seedlings as explant materials, inoculating them into embryonic callus proliferation medium to induce embryonic callus; The embryonic callus proliferation medium is: 1.0 mg / L-2.0 mg / L 6-BA + 0.5 mg / L-1.0 mg / L NAA or 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 the callus-somatic embryo mixture: The embryonic callus obtained in step S1 further develops into a callus-somatic embryo mixture. The callus-somatic embryo mixture obtained in step S1 is cut into small pieces suitable for inoculation, and then inoculated into a callus-somatic embryo mixture proliferation medium for proliferation induction to obtain clustered shoots. Step S3: The clustered buds induced in step S2 are divided into individual plants and then inoculated into a blank MS medium for transitional culture; when the test tube seedlings are 1.5-2.0 cm tall and have 15-20 thick leaves, they are acclimated and transplanted.

2. A method for preserving and commercializing the germplasm of green flower Echeveria according to claim 1, characterized in that: The embryonic callus proliferation medium in step S1 is more preferably 1.5 mg / L-2.0 mg / L 6-BA + 1.0 mg / L 2,4-D.

3. A method for preserving and commercializing the germplasm of green flower Echeveria according to claim 2, characterized in that: The embryonic callus proliferation medium in step S1 is more preferably 1.5 mg / L 6-BA + 1.0 mg / L 2,4-D.

4. The method for preserving and commercializing the germplasm of Echeveria oleifera according to claim 1, wherein: In step S2, the callus-somatic embryo mixture proliferation culture medium is: 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.

5. A method for preserving and commercializing the germplasm of green flower Echeveria according to claim 4, characterized in that: The callus-somatic embryo mixture proliferation medium in step S2 is 1.5 mg / L 6-BA + 0.5 mg / L NAA + 1.0 mg / L 2,4-D.

6. The method for preserving and commercializing the germplasm of Echeveria oleifera according to claim 1, characterized in that: The method further includes step S2.1 between step S2 and step S3: dividing the callus-somatic embryo mixture obtained by proliferation in step S2 into 2-3 seedlings and re-inoculating the seedlings with a small amount of embryonic callus tissue into the culture medium of the callus-somatic embryo mixture proliferation culture medium in step S2 for seedling culture.

7. The method for preserving and commercializing the germplasm of Echeveria oleifera according to claim 1, characterized in that: The "small piece suitable for inoculation" in step S2 is 0.5 cm-1.2 cm×0.5 cm-1.2 cm.

8. The method for preserving and commercializing the germplasm of Echeveria oleifera according to claim 1, characterized in that: During the tissue culture in steps S1-S3, the temperature of the culture chamber was controlled at 22 ± 1°C, the light intensity was 1500-2000 lx, and the illumination time was 10 h / d.

9. The method for preserving and commercializing the germplasm of Echeveria oleifera according to claim 1, wherein: Before step S1, the method further includes the following step S0: selecting well-grown and robust leaves as explants, inoculating them into sterile seedling culture medium, culturing and expanding them to obtain the first batch of sterile seedlings, and obtaining more explant materials from the sterile seedlings.

10. A method for preserving and commercializing the rapid propagation of Echeveria oleifera germplasm according to claim 9, characterized in that: In step S0, the sterile seedling culture medium is: MS + 1.0 mg / L 6-BA + 1.0 mg / L NAA.

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