Dendrobium officinale tetraploid induction method and efficient artificial propagation method thereof

Abstract

The invention relates to the technical field of polyploid induction, and discloses a dendrobium officinale tetraploid induction method and an efficient artificial propagation method thereof.After stem segments with nodes of dendrobium officinale serve as explants and are induced in a colchicine soaking mode to form tetraploids, the induction rate reaches 33.48%, the chimera rate is only 10.37%, and the chimera rate is only 10.37%; the method is obviously superior to a protocorm induction method (plt; 0.05) of the substrate (1); a strategy of'three-step identification-culture medium optimization-double-track proliferation 'is adopted, three-step identification is performed through form-chromosome counting and flow cytometry, regeneration strategy optimization is realized by utilizing an optimized tetraploid proliferation culture medium and simultaneously through'axillary bud-PLBs double-track system', and the final total proliferation coefficient reaches 30.0. The annual yield efficiency is greatly improved, the problem that large-scale production of the traditional artificial induction tetraploid is difficult to realize is solved, and the method has extremely high popularization and application advantages.

Description

Technical Field

[0001] The present invention relates to the technical field of polyploid induction, and particularly relates to a method for inducing tetraploid Dendrobium officinale and an efficient artificial propagation method thereof. Background Art

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

[0003] As the carrier of genetic diversity and the material basis for species evolution research, biological germplasm resources have irreplaceable strategic value in maintaining the stability of the ecosystem and ensuring the sustainable development of humanity. Among them, the germplasm resources of medicinal plants, due to their unique ability to synthesize secondary metabolites, are not only the core elements of the inheritance of traditional Chinese medicine but also the key genetic treasure house for the research and development of modern innovative drugs. However, approximately 4,000 medicinal plant species worldwide are facing the dual threats of habitat fragmentation and over-exploitation, leading to a sharp decline in population genetic diversity, increased inbreeding depression, and a significant decline in the potential for adaptive evolution and other linked ecological crises. More seriously, the phenological disorders and the contraction of suitable habitats caused by climate change have made species with a weak genetic basis show significant environmental adaptation obstacles, which pose a major scientific challenge to the protection and sustainable utilization of medicinal plant germplasm resources.

[0004] Dendrobium officinale Kimura et Migo, also known as Heijiecao, is a perennial epiphytic herb of the genus Dendrobium in the family Orchidaceae; it grows on semi-shady and humid mountain rocks at an altitude of 1,600 - 2,100 m, prefers a warm and humid climate, is not cold-tolerant, and the air humidity is 60% - 80%, and the most suitable temperature during the growth season is 20 - 25°C; the genus Dendrobium is one of the most species-rich genera in the Orchidaceae family, with approximately 1,500 species globally, and its genetic information library contains gene resources for key traits such as stress resistance and the synthesis of medicinal components. However, the wild resources of Dendrobium officinale are on the verge of exhaustion due to long-term over-exploitation, and the existing populations are facing problems such as a decline in genetic diversity, a decrease in stress resistance, and instability of medicinal components. The existing industrial cultivation mainly relies on ramet propagation or tissue culture rapid propagation, resulting in an increasing homogenization of germplasm genetics and an increasingly significant phenomenon of variety degradation, seriously restricting its sustainable development and utilization.

[0005] As an important driving force in plant evolution, polyploidization triggers systematic changes in cell structure and physiological functions through chromosome doubling, demonstrating unique advantages in the development of medicinal resources and crop improvement. Existing research shows that there is a significant correlation between the morphological improvement of vegetative organs and the accumulation of secondary metabolites in polyploid plants. Stress resistance studies have confirmed that polyploid plants enhance their environmental adaptability through multi-level adaptive evolution. These adaptive evolutionary characteristics provide an important theoretical basis for stress-resistant breeding. However, the application of polyploid plants still faces key limitations. Biological limitations such as reduced bioaccumulation rate, gene regulation disorders, and tissue morphological variations are inherently antagonistic to the formation of advantageous traits, becoming the main obstacles to the breakthrough of polyploid breeding technology. Nevertheless, ploidy breeding remains an important technical path for germplasm innovation, and its academic value and theoretical significance cannot be questioned.

[0006] Compared with conventional breeding, polyploid breeding can break through the interspecific hybridization barrier by doubling the chromosome set, directly inducing the generation of new polyploid germplasms, and reconstructing the genomic dosage effect and interaction relationship at the cytological level. This genetic remodeling can not only significantly expand the genetic variation spectrum of germplasms, but also activate secondary metabolic pathways through epigenetic regulation, providing a new approach for the targeted improvement of the active ingredients of medicinal plants. More importantly, the characteristics such as organ gigantism and enhanced stress resistance commonly exhibited by polyploid plants can effectively compensate for the ecological adaptability lost by wild populations due to genetic erosion, providing an innovative solution for the ex-situ conservation and population rejuvenation of endangered medicinal plants.

[0007] In Dendrobium officinale, protocorms are mostly used as experimental materials in polyploid induction research, but multiple technical challenges are faced in actual operation. First, the cell division activity of protocorms is relatively low and the degree of differentiation is relatively high, resulting in limited induction efficiency, and a large number of samples need to be processed to obtain effective variants. Second, its dense tissue structure significantly hinders the uniform penetration of agents, and chimeras are easily formed. Third, during the colchicine treatment process, material death is often caused by toxic effects, and phenotypic changes such as enlarged stomata occur in some treatment groups without accompanying chromosome ploidy changes, resulting in a lack of stable correlation between morphological indicators and genetic variations, and easy phenotypic misjudgment, which is not conducive to the judgment of tetraploid plants with long-term stable inheritance. Summary of the Invention

[0008] The object of the present invention is to provide a method for inducing tetraploid Dendrobium officinale and its efficient artificial propagation method, aiming at the technical problems existing in the induction of polyploid Dendrobium officinale at present, such as low induction rate, easy formation of chimeras, and genetic instability. By innovatively selecting stem segments as the induction materials, based on the characteristics of active cell division in the meristematic tissues of stem nodes, and combining the advantages of simple morphological structure of stem segments and easy penetration of medicaments, the chromosome doubling efficiency is significantly improved. Through the establishment of a three-step identification system including morphological observation (stomatal characteristic analysis), flow cytometry detection (DNA content determination), and chromosome counting verification, tetraploid plants with genetic stability are successfully obtained, effectively solving the problems of high chimera formation rate and phenotypic misjudgment in the traditional induction process, providing a repeatable technical path for polyploid breeding of medicinal plants in the genus Dendrobium, and laying an experimental foundation for analyzing the interaction mechanism between organ development and ploidy regulation.

[0009] The technical solution of the present invention is as follows:

[0010] A method for inducing tetraploid Dendrobium officinale, comprising the following steps:

[0011] Step (1): Obtaining explants: For diploid Dendrobium officinale sterile test-tube seedlings, on a super-clean workbench, use a scalpel to cut the test-tube seedlings into stem segments with nodes about 2.0 - 3.0 cm long, and inoculate them into the diploid Dendrobium officinale proliferation medium (hereinafter referred to as Medium A);

[0012] Step (2): Tetraploid induction: Take stem segments with nodes from the diploid sterile seedlings propagated for 10 - 15 days in step (1), soak the stem segments with nodes in colchicine with a concentration of 0.05% for more than 168 h; after soaking, rinse the stem segment materials with sterile water 3 times, each time not less than 3 min; then inoculate them into Medium A;

[0013] Step (3): Polyploid identification and subculture: Use chromosome counting and flow cytometry methods to identify the polyploidy of the induced plants; the successfully identified plants are subjected to continuous subculture; stable tetraploid plants are obtained.

[0014] According to a preferred embodiment, the soaking duration is 228 h.

[0015] According to a preferred embodiment, the formula of the diploid proliferation medium (Medium A) is: 1 / 3MS + 15 g / L sucrose + 60 g / L banana + 40 g / L potato + 1.0 mg / L - 1.5 mg / L NAA + 1.0 mg / L - 1.5 mg / L KT + 0.1 mg / L - 0.5 mg / L 6 - BA + 1.0 g / L - 1.5 g / L AC.

[0016] According to a preferred embodiment, the formula of medium A is: 1 / 3 MS + 15 g / L sucrose + 60 g / L banana + 40 g / L potato + 1.0 mg / L NAA + 1.0 mg / L KT + 0.1 mg / L 6-BA + 1.0 g / L AC.

[0017] On the other hand, the present invention provides a method for efficient artificial propagation of the tetraploid Dendrobium officinale as described above, comprising the following steps:

[0018] Step (1): Induce and culture the stem segments to be cultured and the protocorm-like bodies simultaneously in a tetraploid proliferation medium for proliferation.

[0019] According to a preferred embodiment, step (1) specifically includes the following sub-steps:

[0020] Step (1.1): Axillary bud proliferation: Take the stem segments with nodes of the selected stable tetraploid plants and inoculate them into the medium with the optimal factor combination to induce cluster buds and rooting;

[0021] Efficient induction of cluster buds and rooting can be achieved through one medium, reducing the difficulty of propagation and simplifying the operation; the proliferation coefficient reaches 10.11.

[0022] Step (1.2): Protocorm-like body proliferation: Cut the protocorm-like body mass at the contact between the cut of the tetraploid plant and the medium into pieces of 1.0×1.0 cm and transfer them to the tetraploid proliferation medium; after culturing until adventitious roots and cluster buds are produced, cut the cluster buds into clusters of 4 - 5 plants and transfer them to a new tetraploid proliferation medium; after culturing until the small buds have a complete plant morphology, with roots, stems, and leaves clearly visible, divide a cluster into single plants and inoculate them into a new tetraploid proliferation medium for culture; the proliferation coefficient reaches 20.0;

[0023] According to a preferred embodiment, the formula of the tetraploid proliferation medium in step (1) is: 0.5 mg / L - 2.5 mg / L NAA + 0.5 mg / L - 2.5 mg / L KT + 0.5 mg / L - 1.5 mg / L 6-BA + 1.0 mg / L - 1.5 mg / L IBA;

[0024] According to a preferred embodiment, the formulation of the tetraploid proliferation medium is: 2.5 mg / L NAA + 0.5 mg / L KT + 1.5 mg / L 6-BA + 1.5 mg / L IBA; or 0.5 mg / L NAA + 1.5 mg / L KT + 1.5 mg / L 6-BA + 1.5 mg / L IBA; or 1.0 mg / L NAA + 0.5 mg / L KT + 1.0 mg / L 6-BA + 1.5 mg / L IBA; or 1.5 mg / L NAA + 1.0 mg / L KT + 2.5 mg / L 6-BA + 1.5 mg / L IBA; or 2.5 mg / L NAA + 2.5 mg / L KT + 0.5 mg / L 6-BA + 1.5 mg / L IBA.

[0025] According to a preferred embodiment, the method for highly efficient artificial propagation of the Dendrobium officinale tetraploid further comprises the following steps:

[0026] Step (2) Acclimatization and seedling hardening:

[0027] Step (3) Transplanting: After seedling hardening, transplant to the treated crushed pine bark.

[0028] Compared with the existing technology, the beneficial effects of the present invention are as follows:

[0029] 1. A method for inducing Dendrobium officinale tetraploid uses nodal stem segments as explants instead of protocorms. The tetraploid induction rate reaches 33.48%, and the chimera rate is only 10.37% (about 60% lower than the previously reported protocorm induction method), which is significantly superior to the protocorm induction method (p < 0.05); the 228 h treatment group achieves the best balance between survival rate (6.78%) and tetraploid induction rate (33.48%), indicating that the meristematic cells of the stem segments precisely capture the colchicine action window through a cycle synchronization of about 9.5 d; its efficiency advantage stems from the uniform permeability and cycle synchronization of the stem meristem. This characteristic is difficult to achieve in seed induction due to cell cycle heterogeneity.

[0030] 2. A method for inducing Dendrobium officinale tetraploid has a single-induced tetraploid proliferation coefficient of 10.11, and only one tetraploid proliferation medium is required to achieve the integration of proliferation and rooting culture; specifically induced protocorm-like bodies (PLBs) are formed at the cut of the stem segments, and the proliferation coefficient reaches 20.0. Combining axillary bud proliferation to form a "dual-track regeneration" system, high-efficiency clonal propagation is achieved through the axillary bud-PLBs dual-track proliferation system, which may further weaken its dependence on sexual reproduction and form a unique "asexual-dominated" adaptation strategy, and the total proliferation coefficient can reach more than 30.0;

[0031] 3. A method for inducing tetraploid of Dendrobium officinale. After acclimatization of test-tube seedlings, the transplanting survival rate can reach 100%, and the plant height is 28 cm after 360 days, indicating that the induced tetraploid retains and even enhances the ecological adaptability of the wild type; "Three-step identification - medium optimization - dual-track proliferation", breaking through the efficiency limit of polyploid through the synchrony of meristem cycle;

[0032] 4. An efficient artificial propagation system for tetraploid of Dendrobium officinale. The main effect value of IBA on the proliferation coefficient of tetraploid Dendrobium officinale (R = 2.135) is significantly higher than that of other hormones, revealing the unique dependence of the polyploid rapid propagation system on auxin substances. In the optimal combination, the synergistic effect of high-concentration NAA and IBA may drive axillary bud formation and adventitious root formation synchronously by promoting vascular bundle differentiation, and its proliferation coefficient reaches 10.11, which is 133.7% higher than that of the diploid medium (medium A). This discovery breaks through the traditional paradigm that the rapid propagation of Orchidaceae plants depends on cytokinin (such as 6-BA) dominance, indicating that polyploidization may enhance the response efficiency to exogenous auxin by changing the endogenous hormone metabolism system. The peak effect of IBA concentration is reached at 1.5 mg / L (proliferation coefficient 9.89), while the proliferation is significantly inhibited when the concentration is increased to 2.5 mg / L, showing a typical biphasic dose effect, which is consistent with the rule of "low promotion and high inhibition" of auxin in the rapid propagation of tetraploid Salvia miltiorrhiza. It is speculated that the effective response range of polyploid cells to hormone sensitivity is reduced.

[0033] 5. An efficient artificial propagation system for tetraploid of Dendrobium officinale. The incidence of specifically induced protocorm-like bodies (PLBs) at the cut of the material (20.0%) indicates a significant correlation between tetraploidization and regeneration ability. Mechanical cutting may activate the somatic reprogramming process by releasing endogenous damage signal molecules (such as jasmonic acid substances) and coordinating the genomic redundancy characteristics of polyploid cells, promoting the PLB proliferation coefficient to reach 20.0. This synergistic effect of damage response provides a new way for efficient regeneration. Further analysis shows that tetraploid plants optimize the regeneration strategy through the "axillary bud - PLB dual-track system": the axillary bud proliferation pathway ensures genetic stability, while the PLB occurrence pathway provides emergency propagation ability. The two work together to greatly improve the annual production efficiency, fundamentally solving the problem that it is difficult to achieve large-scale production in traditional artificial induction of tetraploid, and providing a technical paradigm for ploidy breeding of medicinal plants. The tetraploid genotype may obtain a significant advantage in branch regeneration by enhancing the ability of PLB occurrence. The survival rate of tetraploid plants remains 100% after domestication and transplantation, and there is no significant difference in the internode number between PLB-derived seedlings and axillary bud seedlings (p > 0.05), proving the reliability of its regeneration system. Description of the Drawings

[0034] Figure 1 For the results of flow cytometry analysis (left - control diploid plants; right - tetraploid plants);

[0035] Figure 2 For root tip chromosome squash; in the figure, A, B, and C are 2n = 2x = 38; D, E, and F are 2n = 4x = 76.

[0036] Scale bar = 10 μm;

[0037] Figure 3 For the proliferation culture of tetraploid stem segments in medium A; in the figure, A is the material cultured for 20 d; B is the growth condition after 40 d; C is the growth condition after 60 d; D is the growth condition after 80 d; Scale bar = 2 cm;

[0038] Figure 4 For the main effect plot with the proliferation coefficient as a parameter based on the orthogonal results;

[0039] Figure 5 For the integrated culture of proliferation and rooting of tetraploid plants; in the figure, A and B are the growth conditions after 20 d; C and D are the growth conditions after 40 d; E and F are the growth conditions after 60 d; G and H are the growth conditions after 80 d; I and J are the growth conditions after 100 d; K and L are the growth conditions after 120 d;

[0040] Figure 6 For the occurrence, development, and plantlet formation of protocorm-like bodies; in the figure, A is the growth condition after 100 d; B is the protocorm-like body mass; C is a single protocorm-like body; D is the cluster buds; E is the single bud and double buds; F is the small plant. Note: The part pointed by the red arrow is the protocorm-like body.

[0041] Figure 7 For the proliferation of protocorm-like bodies; in the figure, A is the material cultured for 15 d; B is the growth condition after 30 d; C is the growth condition after 45 d; D is the growth condition after 60 d. Scale bar = 2 cm;

[0042] Figure 8 For the proliferation and development of buds; in the figure, A is the material cultured for 15 d; B is the growth condition after 30 d; C is the growth condition after 45 d; D is the growth condition after 60 d. Scale bar = 2 cm. Note: The part pointed by the red arrow is the plant.

[0043] Figure 9 For the acclimatization and transplantation process; in the figure, A is the test-tube plantlet just transplanted into the substrate; B is the test-tube plantlet after 120 d of transplantation; C is the test-tube plantlet after 240 d of transplantation; D is the plant after 360 d of transplantation. Scale bar = 2 cm.

[0044] Figure 10 For the morphological comparison between diploid and tetraploid, in the figure, A is diploid and tetraploid; B is the plantlet outside the bottle; C is the leaf; D and E are the flowers. Note: The diploid is on the left and the tetraploid is on the right in the figure;

[0045] Figure 11Observation diagrams of cross-sections of leaves and stems; in the figures, A and B are longitudinal microscopic diagrams of leaves (A is tetraploid, B is diploid), scale bar = 500 μm; C and D are transverse microscopic diagrams of stems (C is diploid, D is tetraploid); (a - epidermis, b - mesophyll tissue, c - vascular bundle; 1 - leaf sheath, 2 - leaf sheath vascular bundle, 3 - epidermis, 4 - cortex, 5 - vascular bundle, 6 - pith), scale bar = 2000 μm;

[0046] Figure 12 Stomatal comparison; in the figures, A and B are stomata of diploids; C and D are stomata of tetraploids; scale bars for A and C = 100 μm; scale bars for B and D = 50 μm. Detailed implementation manners

[0047] 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 made 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 all be covered within the scope of the present invention. For those conditions not specified in the examples, they are carried out according to conventional conditions or the conditions recommended by the manufacturer. All reagents or instruments not indicating the manufacturer are conventional products that can be purchased commercially. In order to better illustrate the present invention, numerous specific details are given in the following detailed implementation manners. Those skilled in the art should understand that the present invention can still be implemented without some 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 gist of the present invention.

[0048] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art. Unless otherwise specified, the units used in this specification are all international standard units, and the numerical values and numerical ranges appearing in the present invention should all be understood to include the systematic errors inevitable in industrial production.

[0049] The features and performance of the present invention will be further described in detail below in conjunction with the embodiments.

[0050] Example 1

[0051] I. Materials and methods

[0052] 1.1 Materials

[0053] The fresh stems of Dendrobium officinale were collected from the cultivation base of Xiangxin Agricultural Technology Development Co., Ltd. in Yuxi City, Yunnan Province, China (102°31′25″E, 24°26′22″N, Alt: 1688 m), and were identified as Dendrobium officinale Kimura et Migo by Professor Yu Hong of Yunnan University. A diploid tissue culture system has been successfully established. The research material is the sterile test-tube seedlings of diploid Dendrobium officinale. On the ultra-clean workbench, the test-tube seedlings were cut into about 2.0 - 3.0 cm stem segments with nodes using a scalpel and inoculated into the diploid Dendrobium officinale proliferation medium (1 / 3MS + 15 g / L sucrose + 60 g / L banana + 40 g / L potato + 1.0 mg / L NAA + 1.0 mg / L KT + 0.1 mg / L 6-BA + 1.0 g / L AC, hereinafter referred to as Medium A).

[0054] 1.2 Methods

[0055] The plant growth regulators 6-benzylaminopurine (6-BA), Kinetin (KT), 1-naphthylacetic acid (NAA), 2,4-dichlorophenoxyacetic acid (2,4-D), 3-Indole butyric acid (IBA), sucrose, and agar were all of analytical grade and purchased from Beijing Dingguo Biotechnology Co., Ltd. All media were 1 / 3MS media, with the agar addition amount being 0.47%, and the pH value was adjusted to 5.4 - 5.6. The media were sterilized at 126°C for 30 min in an autoclave and reserved. The colchicine solution was sterilized under the same conditions. Unless otherwise specified, all were mass concentrations; during the experiment, if there was contamination, the materials were promptly re-taken to make up the number of experimental bottles.

[0056] Cultivation conditions: The temperature in the culture room was maintained at 23 ± 2°C, the light was 10 h / d, and the light intensity range was 1800 - 2500 lx.

[0057] 1.2.1 Tetraploid induction

[0058] The stem segments with nodes in Section 1.1 were soaked in 0.05% colchicine for 168, 180, 192, 204, 216, 228, 240, 252, 264, 276, 288, 300 h; after soaking, 15 materials were taken at each time period, rinsed 3 times with sterile water, each time for no less than 3 min. They were inoculated into Medium A, with 3 bottles in each treatment group, 5 materials in each bottle, and repeated 3 times.

[0059] 1.2.2 Chromosome counting and flow cytometry

[0060] (1) Chromosome counting:

[0061] For the materials treated with colchicine, when 3 - 5 adventitious roots grow in the culture flask and the adventitious roots are about 2 - 3 cm long, at 9:00 - 10:00 in the morning, take about 1.0 cm of young root tips, immerse them in 0.05% (w / v) colchicine solution, and place them in the dark at 4°C for 3 h; wash the root tips 3 times with distilled water and transfer them to Carnoy fixative (70% ethanol: glacial acetic acid = 3:1, v / v, freshly prepared), and place them at room temperature for 2 h. Wash with 70% ethanol (absolute ethanol: water = 7:3, v / v) 2 times, and dissociate in 1N HCl for 3 - 6 min; after washing 5 - 6 times with distilled water, soften the root tips in 45% acetic acid for 10 min; take the meristematic zone of the root tips, mash them on a glass slide under a stereomicroscope of model JSZ8, and then stain with a small amount of carbol fuchsin for 15 min; cover the sample with a cover glass and gently flatten it to spread the stained cells; observe the prepared temporary slide under a 100× oil immersion objective of an Olympus BH - 2 microscope and determine the chromosome number.

[0062] Number the first - generation autotetraploid plants (the plants grown after colchicine treatment are the first generation) respectively, and conduct continuous cultivation (the small seedlings with the same number indicate being propagated from the same mother plant). Randomly select 10 plants in each generation, and use the chromosome counting method to determine their chromosome numbers. Stop chromosome counting after continuous cultivation for 5 generations; the screened and confirmed polyploid plants are the polyploid plants with stable inheritance.

[0063] (2) Flow cytometry:

[0064] Mix the sample with fresh leaf tissue (0.5 cm 2 ) of the reference plant (Oryza sativa L.) at a ratio of 1:1 and chop them; put them into 1 mL of nuclear isolation buffer (pH 7.0) (containing 45 mM MgCl2·6H2O, 20 mM MOPS, 30 mM sodium citrate, 1% (w / v) PVP, 0.2% (v / v) Tritonx - 100, 10 mM Na2EDTA) and incubate for 10 min. Filter through a 30 - μm nylon sieve to obtain a nuclear suspension. Add 500 μL of PI (50 mg·mL -1 ) solution to the nuclear suspension, place it in the dark on ice for 0.5 - 1 h, and finally analyze the nuclear suspension with a flow cytometer (BD FAC Scalibur). Use the diploid of Dendrobium officinale as a control, collect 10,000 cell nuclei in each experiment, and control the coefficient of variation (CV) within 5%.

[0065] 1.2.3 Establishment of a rapid propagation system for tetraploid one - step seedling formation

[0066] On the basis of Medium A, NAA, KT, 6-BA, and IBA were selected as factors, and nodal stem segments were used as materials. An L 16 (4 5 ) orthogonal experiment (Table 1) was conducted. There were 3 bottles in each treatment group, and 10 materials were inoculated in each bottle, with 3 replicates. After culturing for 120 days, the proliferation coefficient was statistically analyzed.

[0067] Table 1 One-step seedling formation culture of tetraploid L 16 (4 5 ) Orthogonal experimental design

[0068]

[0069] 1.2.4 Domestication and transplantation of tetraploid test-tube seedlings

[0070] When the plants in the culture bottle grew to 12.0 cm, they were acclimatized at room temperature for 10 days. Then the seedlings were taken out, and the remaining agar solid on the roots was washed off. They were soaked in 0.1% carbendazim for 5 minutes and transplanted into sterilized by high-temperature steaming crushed pine bark (about 1.0×1.0 cm) for cultivation with temperature and humidity maintained. After 60 days, the survival rate and growth status of the seedlings were statistically analyzed.

[0071] 1.2.5 Morphological and cytological observations of diploids and their homologous tetraploids

[0072] The height, stem segment diameter (the 5th - 6th nodes from bottom to top), number of leaves, and leaf area of plants with different ploidy levels (all growing in the same environment) were observed and measured using a vernier caliper. (Measure the leaf of the 5th node from bottom to top of the plant; place the leaf on graph paper to trace the leaf outline, and one small square on the graph paper represents 1.0 mm 2 ). The lower epidermis of the leaves of tetraploid and diploid Dendrobium officinale seedlings was torn off respectively and placed under a microscope to observe the stomatal size and number of stomata. Leaves with the same growth time were selected to prepare paraffin cross-sections of leaves with different ploidy levels, and cell characteristics were observed under an optical microscope. Each ploidy material was repeated 3 times, 5 slides were made each time, and 5 fields of view were observed on each slide.

[0073] 1.2.6 Statistical indicators

[0074] SPSS 27.0 (IBM Corp, Armonk, USA) and Excel (MC Corp, Redmond, USA) software were used to process and analyze the collected data. The tetraploid induction rate was calculated. The numerator was the number of plants that were tetraploid for 5 consecutive generations, and only the numbers of the same serial numbers (1 plant for the same serial number) were added together, without considering the proliferation coefficient in the subsequent 4 generations. And in the subsequent culture, as long as diploid cells were found, they were counted into the chimeras according to the serial numbers.

[0075] Induction rate of autotetraploid (%) = Number of autotetraploids / Total number of survived induced stem segments × 100;

[0076] Chimera rate (%) = Number of chimeras / Total number of survived induced stem segments × 100;

[0077] Protocorm-like body (PLB) induction rate (%) = Number of stem segments producing PLBs / Total number of inoculated stem segments × 100;

[0078] Proliferation coefficient = Number of effective transferred materials / Total number of original inoculations;

[0079] Rooting rate (%) = Number of single seedlings producing adventitious roots / Total number of initial inoculations × 100;

[0080] Acclimatization survival rate (%) = (Number of survived plants / Total number of transplanted plants) × 100;

[0081] Stomatal density = Number of stomata in the field of view / Area of the field of view.

[0082] II. Results

[0083] 2.1 Induction and identification of autotetraploid

[0084] The induction efficiency of autotetraploid in Dendrobium officinale is non-linearly correlated with the treatment duration of colchicine (Table 2). The number of survived materials decreased linearly from 10.89 to 3.33 with the extension of treatment time (168 - 288 h), and the survival rate was zero when the treatment time was ≥ 300 h; the autotetraploid induction rate reached the highest value of 33.48% at 228 h, which was significantly higher than that of other treatment groups in the range of 192 - 264 h (p < 0.05); the chimera rate showed a bimodal distribution and reached the peak value (27.41%) at 204 h and then gradually decreased. The 228 h treatment group showed a unique response pattern, and the autotetraploid rate reached the maximum value when the survival rate dropped to the lowest level (6.78). This phenomenon may be due to the interaction between induction efficiency and colchicine toxicity. The results showed that when the 228 h treatment protocol was adopted, the best balance was achieved between the survival rate and the autotetraploid induction rate, providing an experimental basis for optimizing the treatment parameters.

[0085] Table 2 Effects of different treatment times on autotetraploid induction

[0086]

[0087] Note: Different letters in the same column in the table indicate significant differences at the 5% level; the data represent mean ± standard error (SE), the same below.

[0088] A total of 47 autotetraploids and 14 chimeras were obtained from 61 plants treated with the best treatment group detected by flow cytometry. The histogram of flow cytometer analysis of autotetraploid samples is as Figure 1As shown, the value represented by the abscissa is the fluorescence channel value, and the value represented by the ordinate is the relative value of the measured cell number. Among them, the left peak represents the reference plant (Oryza sativa L.), and the right peak represents the sample plant. By observing the value of the abscissa of the peak, the ploidy of the test material can be clearly judged. The abscissa of the peak value of the diploid plant is approximately around 80 ( Figure 1 - left), while the abscissa of the peak value of its autotetraploid plant is approximately around 160 ( Figure 1 - right), and the abscissa of the peak value of the autotetraploid plant is about twice that of the diploid plant.

[0089] The further somatic chromosome counting results are as Figure 2 shown, showing that the chromosome number of the diploid plant is 2n = 2x = 38 ( Figure 2 - A, B, C), while its autotetraploid is 2n = 4x = 76 ( Figure 2 - D, E, F).

[0090] 2.2 Optimization of the autotetraploid rapid propagation system

[0091] After the materials were inoculated into medium A and cultured for 20 days, the upper axillary buds began to germinate ( Figure 3 - A); after 40 days, the axillary buds grew significantly taller, the leaves unfolded, and adventitious roots began to form at the base ( Figure 3 - B); after 60 days, the axillary buds grew rapidly, the stems thickened, and there were more adventitious roots ( Figure 3 - C); after 80 days, the axillary buds all grew into branches with obvious nodes, the adventitious roots were obvious and well-developed, and the multiplication coefficient was about 4.33 at this time ( Figure 3 - D).

[0092] The results of the orthogonal experiment are shown in Table 3. The range R of the multiplication coefficient IBA > R KT > R 6-BA > R NAA > R Error , indicating that all four factors are effective for the integrated culture of the autotetraploid Dendrobium officinale. From the main effect diagram of the multiplication coefficient ( Figure 4)It can be seen that IBA has a significant effect on the proliferation and rooting of Dendrobium officinale stem segments (p<0.05), while the other three factors have no significant effect on proliferation and rooting (p>0.05). The order of IBA points from high to low in the figure also shows that the most suitable level of IBA for the proliferation and rooting of Dendrobium officinale is level 3 (1.5 mg / L), followed by level 2 (1.0 mg / L), and finally level 1 (0.5 mg / L) and level 4 (2.5 mg / L). Through the average analysis, the best factor combination is A4B1C3D3, that is, the tetraploid proliferation medium is: 2.5 mg / L NAA + 0.5 mg / L KT + 1.5 mg / L 6-BA + 1.5 mg / L IBA.

[0093] Table 3 Integration of proliferation and rooting L 16 (4 5 )Orthogonal experiment results

[0094]

[0095]

[0096] After culturing in the above-selected tetraploid proliferation medium for 20 days, axillary buds appeared on the stem segments ( Figure 5 -A, B); after 40 days, the axillary buds elongated significantly, and secondary axillary buds and white adventitious roots appeared at their bases ( Figure 5 -C, D); after 60 days, the leaves of the primary axillary buds unfolded, and the axillary buds and adventitious roots continued to increase ( Figure 5 -E, F); after 80 days, the primary axillary buds became complete plant-like, the stems became thicker, the leaves were large and extended, and at the same time, more axillary buds and adventitious roots continuously appeared ( Figure 5 -G, H); after 100 days, with the formation of a well-developed adventitious root system, in addition to axillary buds, a large number of tiller buds appeared at the connection between the stem and the root ( Figure 5 -I, J); after 120 days, the growth trend of the plants was extremely vigorous, the stems were thick and strong, the number of leaves increased, the roots also became thick and numerous, and the whole culture was in a cluster shape; at this time, the proliferation coefficient could reach 10.11 ( Figure 5 -K, L).

[0097] Similar to its diploid ancestor, some green nodular masses grew at the contact between the cut of the stem tip of some culture materials and the medium ( Figure 6 -A, B). These masses are composed of multiple approximately spherical structures closely combined. They gather together to form a unique cluster shape. The surfaces of these masses are covered with a layer of delicate white fluff, and the texture is relatively loose. The connection between individual spheres is not very tight, and single small spheres are easily dissected, which is a typical structure of Protocorm-like Bodies (PLBs). Figure 6-C). Cut the PLB clusters into pieces of 1.0×1.0 cm and transfer them to the tetraploid proliferation medium. After 60 days of growth, the PLB clusters develop into multiple shoots, which can be divided into single shoots, double shoots, etc., with adventitious roots ( Figure 6 -D, E); Cut the multiple shoots developed from the PLB clusters into clusters of 4-5 plants per cluster and transfer them to a new tetraploid proliferation medium. After 60 days of growth, they develop into small plants with nodes and adventitious roots ( Figure 6 -F). At this time, the small plants already have a complete plant morphology, and the roots, stems, and leaves are clearly visible. Divide a cluster into single plants and inoculate them into a new medium for cultivation.

[0098] The overall culture process of the aforementioned protocorm-like bodies changes as shown in Figure 7 and Figure 8 . After 15 days of culture, the protocorm-like body (PLB) clusters show a compact structural feature and are accompanied by a progressive increase in volume, showing an improvement in morphological fullness ( Figure 7 -A); At 30 days, continuous proliferation activities lead to a significant increase in the cluster size, and the epidermis differentiates into a dense hairy structure, showing a unique villous surface texture ( Figure 7 -B); After 45 days of culture, the development process shows the appearance of slender filamentous adventitious roots, and at the same time, the meristematic activity is enhanced, and a large number of spherical propagules are produced inside the PLB clusters ( Figure 7 -C); When cultured for 60 days, it can be observed that the population density of PLBs shows exponential growth and there is a phenomenon of cluster fusion; at this time, the proliferation coefficient can reach about 20.0. It should be noted that the primordial shoot apical structure begins to differentiate in a specific meristematic region, marking a clear transition from undifferentiated cell clusters to an ordered plant morphology ( Figure 7 -D).

[0099] After aseptically transferring the shoot clusters (4-5 plants per cluster) to fresh medium, significant morphological changes were observed during the culture process ( Figure 8 -A). At 15 days, the pigmentation of the multiple shoots was significantly enhanced, and at the same time, axillary bud germination occurred at the periphery of the multiple shoots ( Figure 8 -B). By 30 days, continuous proliferation was manifested as an increase in the number of seedlings and vertical elongation growth ( Figure 8 -C). After 60 days of culture, morphogenesis was reflected by the differentiation of nodal structures, and each plant formed 2-3 internodes, marking the transition of test-tube seedlings to autotrophic growth; at this time, the proliferation coefficient was about 20.0 ( Figure 8 -D).

[0100] Breakthroughly, the specific occurrence of PLBs at the cut sites of stem segments reveals the evolutionary association between polyploidization and injury response. Combining with the "dual-track regeneration" system formed by axillary bud proliferation, it provides an adaptive strategy for the regeneration of broken branches in the epiphytic environment. This study has achieved the first full-chain technological innovation of tetraploid Dendrobium officinale from induction, rapid propagation to domestication, providing a theoretical paradigm and practical tool for polyploid breeding of medicinal plants.

[0101] 2.3 Domestication and transplantation results

[0102] When the roots grow to about 3 - 4 cm long, they are domesticated. The rooted seedlings are first placed indoors for 5 days, and then transferred outdoors for 3 days for hardening. During this period, the caps of the glass culture bottles are kept open.

[0103] After hardening, they are transplanted into the treated crushed pine bark, and the survival rate is 100% after 60 days ( Figure 9 -A); after 120 days, the leaves of the plants become significantly larger, the stems become thicker, and the basal buds grow rapidly ( Figure 9 -B); after 240 days, the plants grow significantly taller, and new basal buds appear, starting to show a cluster-like shape ( Figure 9 -C); after 360 days, the complete life cycle is completed, the standard yellow flower morphology is observed in the reproductive growth stage, the final height of the plants is about 28 cm, and the survival rate remains 100% (n = 50) ( Figure 9 -D).

[0104] 2.4 Morphological and anatomical comparison results

[0105] After 12 months of cultivation, both diploid and tetraploid plants have completed the morphological construction of the vegetative growth stage ( Figure 10 -A, B). Quantitative analysis shows (Table 4): The height of the apical branches of tetraploid plants (32.31 cm) is significantly increased by 115.8% compared with that of diploid plants (14.98 cm) (p < 0.05), and the leaf area (735.00 mm 2 ) is increased by 64.6% compared with that of diploid plants (445.11 mm 2 ) (p < 0.01) ( Figure 10 -C). However, there are no statistically significant differences in stem diameter (tetraploid: 5.98 mm vs diploid: 5.05 mm), number of branches (7.45 vs 7.00), and number of leaves (14.60 vs 10.87) between the two (p > 0.05). It is worth noting that the floral organs of tetraploids show significant morphological polymorphism: Although the overall size is larger than that of diploids ( Figure 10 -D), the coverage area of the purple spots at the top of the gynostemium is reduced by 80.3%, and this feature is completely absent in a few individuals ( Figure 10 -E).

[0106] Table 4 Morphological comparison between diploids and tetraploids

[0107]

[0108] Anatomical analysis showed that ( Figure 11 ), the thickness of the mesophyll tissue of the diploid leaves was significantly greater than that of the tetraploid, and the vascular bundle of the tetraploid ( Figure 11 -A) was larger than that of the diploid ( Figure 11 -B), and there was no obvious difference in the epidermis between the two. The microscopic structure of the stem cross-section showed that the cortex thickness of the tetraploid ( Figure 11 -D) was increased compared with that of the diploid ( Figure 11 -C), but the medullary cavities of both showed degenerative characteristics, and there were no obvious differences in other structures. The stomatal characteristics analysis ( Figure 12 ) showed that the stomatal length (67 μm) of the lower epidermis of the tetraploid was increased by 60% compared with that of the diploid (42 μm), while the stomatal density was decreased by 50%.

[0109] The above-described embodiments only represent the specific implementation manners of the present application, and the description thereof is relatively specific and detailed, but it should not be construed as a limitation to the protection scope of the present application. It should be noted that for those of ordinary skill in the art, without departing from the concept of the technical solution of the present application, several modifications and improvements can still be made, and these all belong to the protection scope of the present application.

Claims

1. A method for inducing tetraploid of Dendrobium officinale, characterized in that, It includes the following steps: Step (1): Explant acquisition: For diploid Dendrobium officinale sterile test-tube seedlings, on a clean bench, use a scalpel to cut the test-tube seedlings into about 2.0 - 3.0 cm stem segments with nodes, and inoculate them into the diploid Dendrobium officinale proliferation medium. Step (2): Tetraploid induction: Take stem segments with nodes from the diploid sterile seedlings propagated in step (1) and soak them in colchicine with a concentration of 0.05% for more than 168 h. After soaking, rinse the stem segments with nodes 3 times with sterile water, each time for no less than 3 min. Then inoculate them into the diploid Dendrobium officinale proliferation medium. Step (3): Polyploid identification and subculture: Use chromosome counting and flow cytometry to identify the polyploidy of the induced plants. Continuously subculture the successfully identified plants to obtain stable tetraploid plants.

2. The method for inducing tetraploid of Dendrobium officinale according to claim 1, characterized in that, The stem segments with nodes are soaked in colchicine for 216 h - 276 h.

3. A method for inducing tetraploid Dendrobium officinale according to claim 2, characterized in that, The stem segments with nodes are soaked in colchicine for 228 h - 264 h.

4. A method for inducing tetraploid Dendrobium officinale according to claim 3, characterized in that, The stem segments with nodes are soaked in colchicine for 228 h.

5. A method for inducing tetraploid of Dendrobium officinale according to claim 1, characterized in that, The formula of the diploid Dendrobium officinale proliferation medium is: 1 / 3MS + 15 g / L sucrose + 60 g / L banana + 40 g / L potato + 1.0 mg / L - 1.5 mg / L NAA + 1.0 mg / L - 1.5 mg / L KT + 0.1 mg / L - 0.5 mg / L 6 - BA + 1.0 g / L - 1.5 g / L AC.

6. A method for efficient artificial propagation of tetraploid Dendrobium officinale, characterized in that, It includes the following steps: Step (1) Induce and proliferate the stem segments with nodes or protocorm-like bodies respectively in the tetraploid proliferation medium.

7. A method for highly efficient artificial propagation of tetraploid Dendrobium officinale according to claim 6, characterized in that, Step (1) specifically includes the following sub-steps: Step (1.1): Axillary bud proliferation: Take stem segments with nodes from the stable tetraploid plants obtained by screening and inoculate them into the optimal factor combination medium to induce cluster buds and root formation. Or step (1.2): Protocorm-like body proliferation: Cut the protocorm-like body mass at the contact between the cut of the tetraploid plant and the medium into 1.0×1.0 cm size and transfer it to the tetraploid proliferation medium. After culturing until adventitious roots and cluster buds are produced, cut the cluster buds into clusters of 4 - 5 plants and transfer them to a new tetraploid proliferation medium. After culturing until the small buds have a complete plant form, with clear roots, stems and leaves visible, divide a cluster into single plants and inoculate them into a new tetraploid proliferation medium for culturing to obtain tetraploid plantlets.

8. A method for highly efficient artificial propagation of tetraploid Dendrobium officinale according to claim 6, characterized in that, The formula of the tetraploid proliferation medium is: 0.5 mg / L - 2.5 mg / L NAA + 0.5 mg / L - 2.5 mg / L KT + 0.5 mg / L - 1.5 mg / L 6 - BA + 1.0 mg / L - 1.5 mg / L IBA.

9. The high-efficiency artificial propagation method of Dendrobium officinale tetraploid according to claim 8, characterized in that The formula of the tetraploid proliferation medium is: 2.5 mg / L NAA + 0.5 mg / L KT + 1.5 mg / L 6-BA + 1.5 mg / L IBA; or 0.5 mg / L NAA + 1.5 mg / L KT + 1.5 mg / L 6-BA + 1.5 mg / L IBA; or 1.0 mg / L NAA + 0.5 mg / L KT + 1.0 mg / L 6-BA + 1.5 mg / L IBA; or 1.5 mg / L NAA + 1.0 mg / L KT + 2.5 mg / L 6-BA + 1.5 mg / L IBA; or 2.5 mg / L NAA + 2.5 mg / L KT + 0.5 mg / L 6-BA + 1.5 mg / L IBA.

10. A method for highly efficient induction and artificial propagation of tetraploid Dendrobium officinale, characterized in that, It includes the following steps: Step (1): Tetraploid induction: Perform tetraploid induction according to the tetraploid induction method of Dendrobium officinale as described in any one of claims 1-5. Step (2): Tetraploid propagation: Perform tetraploid propagation according to the high-efficiency artificial propagation method of Dendrobium officinale tetraploid as described in any one of claims 6-9.

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