Dendrobium officinale tetraploid induction method and efficient artificial propagation method thereof

Abstract

The present application relates to the technical field of polyploidy induction, and discloses a Dendrobium officinale four-ploid induction method and an efficient artificial propagation method, wherein a stem section with nodes of Dendrobium officinale is used as an explant, and colchicine is used to induce the formation of four-ploid, and the induction rate reaches 33.48%, and the chimeric rate is only 10.37%, which is significantly better than the original bulb induction method (p<0.05); a strategy of 'three-step identification-culture medium optimization-double track propagation' is used, three-step identification is performed through morphology-chromosome counting and flow cytometry, and the optimized four-ploid propagation culture medium is used to realize the optimization of the regeneration strategy through the 'axillary bud-PLB double track system', and finally the total propagation coefficient reaches 30.0. The annual production efficiency is greatly improved, the problem that the traditional artificial induction of four-ploid is difficult to realize large-scale production is solved, and the method has a very high popularization and application advantage.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of polyploidy induction, and particularly relates to a Dendrobium officinale Kimura et Migo tetraploid induction method and a high-efficiency artificial propagation method thereof. BACKGROUND

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

[0003] Biological germplasm resources, as carriers of genetic diversity and material basis for species evolution research, have irreplaceable strategic value in maintaining ecosystem stability and ensuring human sustainable development. Among them, medicinal plant germplasm resources, due to their unique secondary metabolite synthesis ability, are not only the core elements of traditional Chinese medicine inheritance, but also the key genetic treasure for modern innovative drug research and development. However, about 4000 species of medicinal plants worldwide are facing the dual stress of habitat fragmentation and over-exploitation, leading to a sharp decline in population genetic diversity, intensified inbreeding depression, and significant decline in adaptive evolution potential. More seriously, the phenological disorder and habitat contraction caused by climate change make species with weak genetic foundation show significant environmental adaptation obstacles, which poses a major scientific challenge to the protection and sustainable use 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 rocks at an altitude of 1600-2100m, prefers warm and humid climate, and is not cold-tolerant. The optimal temperature for growth is 20-25℃, and the air humidity is 60%-80%. Dendrobium is one of the most diverse genera in Orchidaceae, with about 1500 species worldwide, and its genetic information database contains genes of key traits such as stress resistance and medicinal ingredient synthesis. However, the wild resources of Dendrobium officinale have been close to exhaustion due to long-term over-exploitation, and the existing populations are facing problems such as genetic diversity decline, stress resistance reduction, and instability of medicinal ingredients. Existing industrial cultivation mainly relies on division propagation or tissue culture, leading to increasing genetic homogenization of germplasm and significant degradation of varieties, which seriously restricts the sustainable development and utilization of Dendrobium officinale.

[0005] Polyploidization, as an important driving force of plant evolution, triggers systematic changes in cell structure and physiological functions through chromosome doubling, and shows unique advantages in the development of medicinal resources and crop improvement. Existing studies have shown that there is a significant correlation between the morphological improvement of vegetative organs and the accumulation of secondary metabolites in polyploid plants. Studies on stress resistance have confirmed that polyploid plants have improved environmental adaptation ability through multi-level adaptive evolution. These adaptive evolution characteristics provide an important theoretical basis for stress resistance breeding. However, the application of polyploid plants still faces key limitations. Biological accumulation rate reduction, gene regulation disorder, and tissue morphology variation are inherent antagonies between these biological limitations and the formation of superior traits, which are the main obstacles to breakthroughs in polyploid breeding technology. However, polyploidy breeding remains an important technical path for germplasm innovation, and its academic value and theoretical significance cannot be doubted.

[0006] Compared with conventional breeding, polyploid breeding can break through the barriers of intergeneric hybridization by chromosome doubling, directly induce new polyploid germplasm, and reconfigure the genome dosage effect and interaction at the cytological level. This genetic remodeling not only significantly expands the genetic variation spectrum of germplasm, but also activates the secondary metabolic pathway through epigenetic regulation, providing a new approach for the targeted improvement of effective components in medicinal plants. More importantly, the organ hypertrophy and enhanced stress resistance exhibited by polyploid plants can effectively compensate for the loss of ecological adaptability due to genetic erosion in wild populations, providing an innovative solution for the ex situ conservation and population rejuvenation of endangered medicinal plants.

[0007] In Dendrobium officinale, polyploid induction studies often use protocorms as experimental materials, but there are many technical challenges in actual operation. First, the low cell division activity and high differentiation degree of protocorms limit the induction efficiency, and a large number of samples need to be processed to obtain effective variants. Second, the dense tissue structure significantly hinders the uniform penetration of reagents, making it easy to form chimeras. Third, the toxic effects of colchicine treatment often cause material death, and some treatment groups show increased stomatal size without accompanying chromosomal ploidy changes, leading to a lack of stable correlation between morphological indicators and genetic variation, which can easily lead to phenotypic misjudgment and is not conducive to identifying long-term stable tetraploid plants. SUMMARY

[0008] The present application aims at the technical problems of low induction rate, easy formation of chimeras and genetic instability in the current induction of Dendrobium officinale polyploids, and provides a Dendrobium officinale tetraploid induction method and an efficient artificial propagation method thereof. Stem segments are innovatively selected as the induction material, based on the characteristics of active cell division of stem node meristem, combined with the advantages of simple morphology and structure of stem segments and easy penetration of reagents, so that the chromosome doubling efficiency is significantly improved. Through the establishment of a three-step identification system of morphological observation (stoma characteristic analysis), flow cytometry detection (DNA content determination) and chromosome counting verification, genetically stable tetraploid plants are successfully obtained, effectively solving the problems of high chimeric formation rate and phenotype misjudgment in the traditional induction process, providing a repeatable technical path for the polyploid breeding of Dendrobium officinale medicinal plants, and laying an experimental foundation for analyzing the interaction mechanism of organ development and ploidy regulation.

[0009] The technical scheme of the present application is as follows:

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

[0011] Step (1): Obtaining explants: diploid Dendrobium officinale sterile test-tube seedlings are cut into about 2.0-3.0 cm nodal stem segments on a clean bench with a surgical knife, and inoculated into a diploid Dendrobium officinale proliferation medium (hereinafter referred to as A medium).

[0012] Step (2): Tetraploid induction: the nodal stem segments are taken from the diploid sterile seedlings cultured for 10-15 days in step (1), and soaked in 0.05% colchicine for more than 168 h; after soaking, the nodal stem segment material is washed with sterile water for 3 times, each time not less than 3 min; and then inoculated into the A medium.

[0013] Step (3): Polyploid identification and subculture: the induced plants are identified by chromosome counting and flow cytometry; the identified plants are continuously subcultured; and stable tetraploid plants are obtained.

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

[0015] According to a preferred embodiment, the formula of the diploid proliferation medium (A 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.

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

[0017] Another aspect of the present application provides an efficient artificial propagation method of the Dendrobium officinale tetraploid as described above, comprising the following steps:

[0018] Step (1) simultaneously culturing the stem segments and the protocorm-like bodies in the tetraploid propagation culture medium for induction and propagation.

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

[0020] Step (1.1) axillary bud propagation: taking the stable tetraploid plant selected to the optimal factor combination culture medium to induce the cluster buds and rooting;

[0021] The induction of the cluster buds and the rooting can be realized by one culture medium, which reduces the difficulty of propagation, simplifies the operation, and the propagation coefficient reaches 10.11.

[0022] Step (1.2) protocorm-like body propagation: cutting the protocorm-like body groups on the cut surface of the tetraploid plant to 1.0*1.0 cm in size and transferring them to the tetraploid propagation culture medium; after culturing to generate the false roots and the cluster buds, cutting the cluster buds to 4-5 plants as a cluster and transferring them to the new tetraploid propagation culture medium; after culturing to generate the small buds with complete plant morphology, the root system, the stem and the leaves are all clear, and the cluster is divided into single plants and inoculated into the new tetraploid propagation culture medium for culture; the propagation coefficient reaches 20.0.

[0023] According to a preferred embodiment, the formula of the tetraploid propagation culture 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 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.

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

[0026] Step (2) acclimatization and hardening-off:

[0027] Step (3) transplanting: transplanting into treated pine bark after hardening-off.

[0028] Compared with the prior art, the present application has the following beneficial effects:

[0029] 1. A Dendrobium officinale Kimura et Migo tetraploid induction method, which uses stem segments with nodes as explants instead of protocorms, and the tetraploid induction rate reaches 33.48%, and the chimeric rate is only 10.37% (about 60% lower than the protocorm induction method reported previously), which is significantly better than the protocorm induction method (p<0.05); the 228h treatment group reaches the optimal balance between the survival rate (6.78%) and the tetraploid induction rate (33.48%), indicating that the stem segment meristem cells accurately capture the colchicine action window through about 9.5d of cycle synchronization; the efficiency advantage is derived from the uniform permeability and cycle synchronization of the stem segment meristem. This characteristic is difficult to achieve in seed induction due to cell cycle heterogeneity.

[0030] 2. A Dendrobium officinale Kimura et Migo tetraploid induction method, which has a single tetraploid proliferation coefficient of 10.11, and only one tetraploid proliferation medium is needed to realize the integration of proliferation, rooting culture, etc.; the stem segment cut surface specifically induces protocorm-like bodies (PLBs), and the proliferation coefficient reaches 20.0; combined with axillary bud proliferation, a "double-track regeneration" system is formed, and through the axillary bud-PLB double-track proliferation system, efficient clonal propagation is realized, which may further weaken its dependence on sexual reproduction, form a unique "asexual dominant" adaptation strategy, and the total proliferation coefficient can reach more than 30.0;

[0031] 3. A method for inducing Dendrobium officinale tetraploid, the survival rate of test-tube seedlings after acclimatization and domestication can reach 100%, and the height of 360 d plants can reach 28 cm, indicating that the induced tetraploid retains or even enhances the ecological adaptability of wild type; "three-step identification-medium optimization-double track propagation", through the cycle synchrony of meristematic tissue, the efficiency of polyploidy is broken through;

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

[0033] 5. A high-efficiency artificial propagation system of Dendrobium officinale tetraploid, the specific induction rate of protocorm-like bodies (PLBs) at the material cut site (20.0%) indicates that there is a significant correlation between tetraploidization and regeneration ability. Mechanical cutting may release endogenous damage signal molecules (such as jasmonates), synergize with the genomic redundancy characteristics of polyploid cells, activate somatic cell reprogramming, and promote the PLBs propagation coefficient to 20.0. This damage response enhancement provides a new way for efficient regeneration. Further analysis shows that tetraploid plants optimize the regeneration strategy through "axillary bud-PLBs double track system": the axillary bud propagation pathway ensures genetic stability, while the PLBs occurrence pathway provides emergency propagation capacity, and the combination of the two greatly improves annual production efficiency, fundamentally solving the problem that traditional artificial induction of tetraploid is difficult to realize large-scale production, and providing a technical paradigm for medicinal plant polyploidy breeding. The tetraploid genotype may obtain significant broken branch regeneration advantage by enhancing the PLBs occurrence ability, and the tetraploid plants still maintain 100% survival rate after domestication and transplantation, and the internode number of PLBs derived seedlings has no significant difference (p>0.05) with that of axillary bud seedlings, proving the reliability of its regeneration system. BRIEF DESCRIPTION OF DRAWINGS

[0034] Figure 1 Flow cytometry analysis results (left-diploid control plants; right-tetraploid plants);

[0035] Figure 2 Root tip squashed for chromosome staining; A, B, C, 2n = 2x = 38; D, E, F, 2n = 4x = 76.

[0036] Scale bar = 10 μm;

[0037] Figure 3 Tetraploid stem segments were proliferated in A medium; A, 20 d of culture; B, 40 d of growth; C, 60 d of growth; D, 80 d of growth; scale bar = 2 cm.

[0038] Figure 4 Main effect plot with proliferation coefficient as parameter based on orthogonal results;

[0039] Figure 5 Tetraploid plant proliferation and rooting integrated culture; A, B, 20 d of growth; C, D, 40 d of growth; E, F, 60 d of growth; G, H, 80 d of growth; I, J, 100 d of growth; K, L, 120 d of growth.

[0040] Figure 6 Pro-embryo-like development, development and seedling; A, 100 d of growth; B, pro-embryo-like mass; C, single pro-embryo-like; D, cluster bud; E, single bud, double bud; F, small plant. Note: the red arrow points to the pro-embryo-like.

[0041] Figure 7 Pro-embryo-like proliferation; A, 15 d of culture; B, 30 d of growth; C, 45 d of growth; D, 60 d of growth. Scale bar = 2 cm.

[0042] Figure 8 Bud proliferation and development; A, 15 d of culture; B, 30 d of growth; C, 45 d of growth; D, 60 d of growth. Scale bar = 2 cm. Note: the red arrow points to the plant.

[0043] Figure 9 Acclimatization and transplanting process; A, test-tube seedling just transplanted into substrate; B, test-tube seedling 120 d after transplanting; C, test-tube seedling 240 d after transplanting; D, plant 360 d after transplanting. Scale bar = 2 cm.

[0044] Figure 10 Diploid and tetraploid morphological comparison; A, diploid, tetraploid; B, off-vessel seedling; C, leaf; D, E, flower. Note: the left side of the figure is diploid and the right side is tetraploid.

[0045] Figure 11Figure 1. Leaf and stem cross-sections; A, B are longitudinal leaf micrographs (A tetraploid, B diploid), scale bar = 500 μm; C, D are cross-sections of stems (C diploid, D tetraploid); (a-epidermis, b- mesophyll tissue, c-vessel; 1-leaf sheath, 2-leaf sheath vessel, 3-epidermis, 4-cortex, 5-vessel, 6-pith), scale bar = 2000 μm;

[0046] Figure 12 Stomata comparison; A, B are diploid stomata; C, D are tetraploid stomata; A, C scale bar = 100 μm; B, D scale bar = 50 μm. DETAILED DESCRIPTION

[0047] The specific examples listed in the present application are only as examples of the present application, and the present application is not limited to the specific examples described below. Any equivalent modifications and substitutions of the examples described below by those skilled in the art are also within the scope of the present application. Therefore, equivalent transformations and modifications made without departing from the spirit and scope of the present application should be encompassed within the scope of the present application. The specific conditions not mentioned in the examples are carried out according to the conventional conditions or the conditions suggested by the manufacturers. All reagents or instruments not mentioned by the manufacturers are conventional products available in the market. In order to better illustrate the present application, numerous specific details are given in the following detailed description of the embodiments. Those skilled in the art should understand that the present application can be implemented without some specific details. In some other embodiments, methods, means, instruments and steps well known to those skilled in the art are not described in detail in order to highlight the main idea of the present application.

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

[0049] The features and properties of the present application are further described in detail below in connection with the examples.

[0050] Example 1

[0051] I. Materials and Methods

[0052] 1.1 Materials

[0053] Fresh Dendrobium officinale Kimura et Migo was collected from the cultivation base of Xiangxin Agricultural Technology Development Co., Ltd. in Yuxi City, Yunnan Province (102°31'25"E, 24°26'22"N, Alt: 1688 m), and 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 aseptic test-tube seedling of diploid Dendrobium officinale. In the clean bench, the test-tube seedling was cut into about 2.0-3.0 cm stem segments with a scalpel, and inoculated in the diploid Dendrobium officinale proliferation medium (1 / 3MS + sucrose 15 g / L + banana 60 g / L + potato 40 g / L + 1.0 mg / L NAA + 1.0 mg / L KT + 0.1 mg / L 6-BA + AC 1.0 g / L, hereinafter referred to as A medium).

[0054] 1.2 Method

[0055] Plant growth regulators 6-benzylaminopurine (6-BA), Kinetin (KT), 1-naphthylacetic acid (NAA), 2,4-dichlorophenoxyacetic acid (2,4-D), and 3-Indole butyric acid (IBA) were all analytical pure and purchased from Beijing Dingguo Biological Technology Co., Ltd. The medium was 1 / 3MS medium, and the agar addition amount was 0.47%, and the pH value was adjusted to 5.4-5.6. The medium was sterilized in a high-pressure sterilization pot at 126℃ for 30 min, and the colchicine solution was sterilized under the same conditions. Unless otherwise specified, the mass concentration was used. If there was pollution during the experiment, the experimental bottle number was supplemented in time.

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

[0057] 1.2.1 Tetraploid induction

[0058] The stem segments in section 1.1 were soaked in 0.05% colchicine for 168, 180, 192, 204, 216, 228, 240, 252, 264, 276, 288, and 300 hours. After soaking, 15 materials were taken from each time period, and all were washed with sterile water for 3 times, each time not less than 3 min. Inoculate in A medium, 3 bottles for each treatment group, 5 materials per bottle, repeated 3 times.

[0059] 1.2.2 Chromosome counting and flow cytometry

[0060] (1) Chromosome counting:

[0061] The material treated by colchicine grew 3-5 adventitious roots in the culture bottle. When the adventitious roots were about 2-3 cm long, the tender root tips of about 1.0 cm were taken between 9:00 and 10:00 in the morning, immersed in 0.05% (w / v) colchicine solution, and placed at 4°C in the dark for 3 h; the root tips were washed with distilled water for 3 times, and transferred to Carnoy fixative (70% ethanol: glacial acetic acid = 3:1, v / v, prepared fresh) at room temperature for 2 h. Washed with 70% ethanol (absolute ethanol: water = 7:3, v / v) for 2 times, and dissociated in 1N HCl for 3-6 min; after washing with distilled water for 5-6 times, the root tips were softened in 45% acetic acid for 10 min; the root tip meristem was crushed on a glass slide under a JSZ8 type body microscope, then stained with a small amount of carmine for 15 min; the sample was covered with a cover glass, and gently flattened to spread the stained cells; the prepared temporary slide was observed under an Olympus BH-2 microscope at 100x oil immersion, and the number of chromosomes was determined.

[0062] The first generation of homologous tetraploid plants (the plants grown after colchicine treatment are the first generation) were numbered respectively, and continuously cultured (the seedlings with the same number represent the propagation from the same mother plant); 10 plants were randomly selected from each generation, and the number of chromosomes was determined by chromosome counting; the chromosome counting was stopped after 5 generations of continuous culture; the screened and confirmed polyploid plants were the stably inherited polyploid plants.

[0063] (2) Flow cytometry:

[0064] The sample was mixed with fresh leaf tissue (0.5 cm 2 ) of the reference plant (Oryza sativa L.) at 1:1, and chopped; it was incubated in 1 mL 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) Triton x-100, and 10 mM Na2EDTA for 10 min, filtered through a 30 μm nylon screen to obtain a nuclear suspension, 500 μL PI (50 mg·mL -1 ) solution was added to the nuclear suspension, and placed on ice in the dark for 0.5-1 h; finally, the nuclear suspension was analyzed by flow cytometry (BD FAC Scalibur), with Dendrobium officinale diploid as the control; 10,000 cell nuclei were collected for each test, and the coefficient of variation (CV) was controlled within 5%.

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

[0066] On the basis of A culture medium, NAA, KT, 6-BA, IBA were selected as factors, with stem segments as materials, L 16 (4 5 ) orthogonal test (Table 1), 3 bottles for each treatment group, 10 materials for each bottle, repeated 3 times; after 120d culture, the proliferation coefficient was counted.

[0067] Table 1 One-step seedling culture 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 grow to 12.0 cm, they are acclimated at room temperature for 10d, then the seedlings are taken out, the seedlings are taken out, the agar solid remaining on the roots is washed off, and they are soaked in 0.1% carbendazim for 5min, then transplanted into crushed pine bark (about 1.0x1.0cm) that has been sterilized by high-temperature steaming and cultured in a humid environment, and after 60d, the survival rate and growth status of the seedlings are counted.

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

[0072] The height, stem diameter (5-6 nodes from the bottom to the top), leaf number, and leaf area (measuring the 5th node leaf from the bottom to the top; placing the leaf on the coordinate paper to outline the leaf contour, and one small square on the coordinate paper represents 1.0mm 2 ) of plants of different ploidy (all grown in the same environment) were observed and measured with a vernier caliper. The lower epidermis of D. candidum tetraploid seedlings and diploid seedlings was torn off and observed under a microscope to measure the size and number of stomata. Leaves of the same growth time were selected to prepare paraffin cross sections of leaves of different ploidy, and the cell characteristics were observed under an optical microscope. Each ploidy material was repeated 3 times, and 5 slides were prepared each time, and 5 fields were observed for each slide.

[0073] 1.2.6 Statistical indicators

[0074] The collected data were processed and analyzed using SPSS 27.0 (IBM Corp, Armonk, USA) and Excel (MC Corp, Redmond, USA) software. The tetraploid induction rate was calculated, and the number of tetraploid plants in the continuous 5 generations was added, and the proliferation coefficient of the subsequent 4 generations was not counted. In the subsequent culture, as long as diploid cells were found, they were counted as chimeras.

[0075] Induction rate of autotetraploid (%) = number of autotetraploid / total number of survival explants x 100;

[0076] Chimera rate (%) = number of chimera / total number of survival explants x 100;

[0077] Protocorm-like body rate (%) = number of explants producing protocorm-like bodies / total number of inoculated explants x 100;

[0078] Proliferation coefficient = number of effective transferred materials / total number of original inoculation;

[0079] Rooting rate (%) = number of single plant producing adventitious roots / total number of initial inoculation x 100;

[0080] Acclimatization survival rate (%) = (number of survival plants / total number of transplanted plants) x 100;

[0081] Stomatal density = number of stomata in a field / area of the field.

[0082] II. Results

[0083] 2.1 Induction and identification of autotetraploid

[0084] The induction efficiency of autotetraploid in D. candidum was nonlinearly correlated with the duration of colchicine treatment (Table 2). The survival number of explants 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 longer than 300 h. The highest induction rate of autotetraploid (33.48%) was observed 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, with the highest value (27.41%) at 204 h and then gradually decreased. The treatment group at 228 h showed a unique response pattern, with the lowest survival rate (6.78) and the highest induction rate of autotetraploid. This phenomenon might be caused by the interaction between the induction efficiency and the toxicity of colchicine. The results showed that the best balance between the survival rate and the induction rate of autotetraploid was achieved at 228 h, which provided experimental evidence for optimizing the treatment parameters.

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

[0086]

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

[0088] Among the 61 plants treated by the best treatment group, 47 autotetraploids and 14 chimeras were obtained by flow cytometry. The flow cytometry analysis histogram of autotetraploid samples is shown in Fig. 2. Figure 1As shown in the figure, 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; wherein the left peak represents the reference plant (Oryza sativa L.), and the right peak represents the sample plant. By observing the value of the peak abscissa, the ploidy of the test material can be clearly judged. The peak abscissa of the diploid plant is about 80( Figure 1 -Left), and the peak abscissa of the homologous tetraploid plant is about 160( Figure 1 -Right), and the peak abscissa of the homologous tetraploid plant is about 2 times that of the diploid plant.

[0089] Further somatic chromosome counting results are shown in Figure 2 , showing that the chromosome number of the diploid plant is 2n = 2x = 38( Figure 2 -A, B, C), and that of the homologous tetraploid is 2n = 4x = 76( Figure 2 -D, E, F).

[0090] 2.2 Optimization of tetraploid rapid propagation system

[0091] After the material is introduced into A medium and cultured for 20d, the high axillary bud begins to germinate( Figure 3 -A); after 40d, the axillary bud grows obviously, the leaf expands, and the base begins to produce adventitious roots( Figure 3 -B); after 60d, the axillary bud grows rapidly, the stem becomes thick, and more adventitious roots are produced( Figure 3 -C); after 80d, the axillary bud grows into a branch with obvious nodes, and the adventitious roots are obvious and well developed, and the proliferation coefficient is about 4.33( Figure 3 -D).

[0092] The orthogonal experiment results are shown in Table 3, and the range R IBA > of the proliferation coefficient KT > is 2.48 6-BA >, 2.48 NAA >, 2.48 Error , indicating that the four factors are effective for the integrated culture of D. candidum tetraploid, and the main effect diagram of the proliferation coefficient( Figure 4)It can be seen that IBA has a significant effect on the proliferation and rooting of D. candidum stem segments (p<0.05), and the other three factors have no significant effect on the proliferation and rooting (p>0.05). The order of IBA from high to low also shows that the most suitable IBA for the proliferation and rooting of D. candidum 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 average analysis, the best factor combination is A4B1C3D3, i.e., 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 Proliferation and rooting integration L 16 (4 5 )Orthogonal experiment results

[0094]

[0095]

[0096] After 20 days of culture in the above-mentioned tetraploid proliferation medium, axillary buds appeared on the stem segments ( Figure 5 -A, B); after 40 days, the axillary buds obviously elongated, secondary axillary buds appeared at their base, and white adventitious roots appeared ( Figure 5 -C, D); after 60 days, the primary axillary bud leaves unfolded, and the axillary buds and adventitious roots continued to increase ( Figure 5 -E, F); after 80 days, the primary axillary buds had become complete plantlets, the stems became thick, the leaves were large and stretched out, and more axillary buds and adventitious roots continuously appeared ( Figure 5 -G, H); after 100 days, with the formation of developed adventitious root systems, a large number of tiller buds appeared at the connection between the stems and roots, in addition to the axillary buds ( Figure 5 -I, J); after 120 days, the plant growth was extremely vigorous, the stems were thick and strong, the leaf number increased, the root system became thick and numerous, and the entire culture presented 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 cut surface of the stem tips of some of the culture materials, which contacted with the culture medium ( Figure 6 -A, B). These masses were composed of multiple nearly spherical structures that were tightly combined together, forming a unique morphology. The surface of these masses was covered with a layer of fine white velvet, and the texture was relatively loose, and the connection between the individual spheres was not very tight, and the individual small spheres could be easily separated, which was a typical PLBs structure ( Figure 6-C). The protocorm-like bodies were cut into 1.0 x 1.0 cm size and transferred to tetraploid proliferation medium. After 60 days of growth, the protocorm-like bodies developed into clumps of buds, which could be divided into single buds, double buds, etc., with rhizoids Figure 6 -D, E); the clumps of buds developed from the protocorm-like bodies were cut into 4-5 plants per clump and transferred to new tetraploid proliferation medium. After 60 days of growth, the clumps developed into small plants with nodes and adventitious roots Figure 6 -F). At this time, the small plants had a complete plant morphology, with clear root systems, stems, and leaves. A cluster was divided into single plants and inoculated into new medium for culture.

[0098] The overall culture process of the aforementioned protocorm-like bodies is shown in Figure 7 and Figure 8 . After 15 days of culture, the protocorm-like body (PLB) mass exhibited a compact structure and gradual swelling in volume, showing an increase in morphological fullness Figure 7 -A); by 30 days, the continuous proliferation activity led to significant enlargement of the mass, with the epidermis differentiating into a dense hairy structure, exhibiting a unique velvety surface texture Figure 7 -B); after 45 days of culture, the development process showed the appearance of fine filamentous rhizoids, with enhanced meristematic activity, and a large number of spherical propagules were produced within the PLB mass Figure 7 -C); by 60 days of culture, the PLB population density showed exponential growth, with mass fusion phenomena observed. At this time, the proliferation coefficient was about 20.0. Notably, the specific meristematic tissue region began to differentiate into a primitive shoot tip structure, marking a clear transition from undifferentiated cell mass to ordered plant morphology Figure 7 -D).

[0099] After the stem clumps (4-5 plants per clump) were aseptically transferred to fresh medium, a significant morphological transition was observed during the culture process Figure 8 -A). By 15 days, the clump pigmentation significantly enhanced, and axillary bud emergence was observed at the periphery of the clumps Figure 8 -B). By 30 days, the continuous proliferation showed an increase in the number of seedlings and vertical elongation growth Figure 8 -C). After 60 days of culture, morphological development was reflected through the differentiation of segmented structures, with 2-3 internodes formed per plant, marking the transition of the test-tube seedlings to independent phototrophic growth. At this time, the proliferation coefficient was about 20.0 Figure 8 -D).

[0100] Breakthroughly, the specific occurrence of PLBs at stem segment incision sites reveals an evolutionary link between polyploidization and damage response. Combined with the "dual-track regeneration" system formed by axillary bud proliferation, this provides an adaptive strategy for branch regeneration in epiphytic environments. This study is the first to achieve a complete technological innovation in the entire chain of tetraploidization of Dendrobium officinale, from induction and rapid propagation to domestication, providing a theoretical paradigm and practical tools for polyploid breeding of medicinal plants.

[0101] 2.3 Results of acclimatization and transplantation

[0102] When the roots reach a length of about 3-4 cm, acclimatization should be carried out. The rooted seedlings should first be placed indoors for 5 days, and then moved outdoors for 3 days to harden off. During this period, the caps of the glass culture bottles should be kept open.

[0103] After hardening off, the seedlings were transplanted into treated pine bark chips, and the survival rate was 100% after 60 days. Figure 9 -A); After 120 days, the plant leaves became significantly larger, the stems thickened, and the basal buds grew rapidly. Figure 9 -B); After 240 days, the plants grew significantly taller, accompanied by the appearance of new basal buds, beginning to show a clustered immature form ( Figure 9 -C); After 360 days, the complete life cycle is completed, and standard yellow flower morphology is observed during the reproductive growth stage. The final plant height is about 28cm, and a 100% survival rate is maintained (n=50). Figure 9 -D).

[0104] 2.4 Comparative results of morphology and anatomy

[0105] After 12 months of cultivation, both diploid and tetraploid plants completed the morphological construction of the vegetative growth stage. Figure 10 -A, B). Quantitative analysis showed (Table 4): the terminal branch height of tetraploid plants (32.31 cm) was significantly increased by 115.8% compared with diploid plants (14.98 cm) (p<0.05), and the leaf area (735.00 mm²) was also significantly increased. 2 Compared to diploid (445.11mm) 2 The increase was 64.6% (p<0.01). Figure 10 -C). However, there were no statistically significant differences between the two groups 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) (p>0.05). Notably, the tetraploid floral organs exhibited significant morphological pleomorphism: although the overall size was larger than that of the diploid ( Figure 10 -D), but the purple spot coverage area at the top of its column is reduced by 80.3%, and a few individuals completely lack this feature ( Figure 10 -E).

[0106] Table 4. Morphological Comparison of Diploid and Tetraploid Forms

[0107]

[0108] Anatomical analysis showed ( Figure 11 The leaf mesophyll tissue thickness of diploid leaves is significantly greater than that of tetraploid leaves. Figure 11 -A) vascular bundles are larger than diploid ( Figure 11 -B), the epidermis of the two is not significantly different. Microstructure of the stem cross section shows that the tetraploid ( Figure 11 -D) Cortical thickness is greater than that of diploid ( Figure 11 -C) increased, but both showed signs of medullary cavity degeneration, with no significant differences in other structures. Stomatal characteristics analysis ( Figure 12 The results showed that the stomatal length of the lower epidermis of the tetraploid (67 μm) increased by 60% compared with that of the diploid (42 μm), while the stomatal density decreased by 50%.

[0109] 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 inducing Dendrobium candidum tetraploid, characterized in that, Comprising the following steps: Step (1): Obtaining explants: diploid Dendrobium officinale sterile test-tube seedlings are cut into 2.0-3.0 cm stem segments with nodes on a clean bench with a surgical knife, and inoculated into a diploid Dendrobium officinale proliferation medium; Step (2): Tetraploid induction: the stem segments with nodes taken from the diploid sterile seedlings bred in step (1) are soaked in 0.05% colchicine for 228-240 hours; after soaking, the stem segments with nodes are washed with sterile water for 3 times, each time for no less than 3 minutes; then inoculated into a diploid Dendrobium officinale proliferation medium; the formula of the diploid Dendrobium officinale proliferation medium is: 1 / 3 MS + 15 g / L sucrose + 60 g / L banana + 40 g / L potato + 1.0-1.5 mg / L NAA + 1.0-1.5 mg / L KT + 0.1-0.5 mg / L 6-BA + 1.0-1.5 g / L AC; Step (3): Polyploid identification and subculture: the induced plants are identified by chromosome counting and flow cytometry; the identified plants are continuously subcultured; and stable tetraploid plants are obtained.

2. The method of claim 1, wherein the method is characterized by, The stem segments with nodes are soaked in colchicine for 228 hours.

3. An artificial propagation method of the Dendrobium candidum tetraploid prepared by the method of claim 1 or 2. Comprising the following steps: Step (1): The stem segments with nodes or protocorm-like bodies are respectively induced and proliferated in a tetraploid proliferation medium; the formula of the tetraploid proliferation medium is: 0.5-2.5 mg / L NAA + 0.5-2.5 mg / L KT + 0.5-1.5 mg / L 6-BA + 1.0-1.5 mg / L IBA; Step (1) specifically comprises the following sub-steps: Step (1.1): Axillary bud proliferation: the stable tetraploid plants screened are taken stem segments with nodes, which are inoculated into a tetraploid proliferation medium to induce cluster buds and rooting; Or step (1.2): Protocorm-like body proliferation: the protocorm-like body groups at the cut and medium contact part of the tetraploid plants are cut into 1.0*1.0 cm size and transferred to a tetraploid proliferation medium; after culturing to produce false roots and cluster buds, the cluster buds are cut into 4-5 plants as a cluster and transferred to a new tetraploid proliferation medium; after culturing to small buds with complete plant morphology, root system, stem and leaves are clearly visible, a cluster is divided into single plants and inoculated into a new tetraploid proliferation medium for culture; and tetraploid plant seedlings are obtained.

4. The method according to claim 3, wherein the method is characterized by, 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 2.5 mg / L NAA + 2.5 mg / L KT + 0.5 mg / L 6-BA + 1.5 mg / L IBA.

5. The method according to claim 3, wherein the method is characterized by, The formula of the tetraploid proliferation medium is replaced by 1.5 mg / L NAA + 1.0 mg / L KT + 2.5 mg / L 6-BA + 1.5 mg / L IBA.

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