Method for inducing jellyfish polyploidy and application thereof

Abstract

The application discloses a jellyfish polyploid induction method and application thereof, and belongs to the technical field of marine ornamental animal breeding. The jellyfish polyploid induction method takes the polyp stage of jellyfish as an induction object, and polyploid jellyfish is obtained by induction treatment through combination of colchicine and dimethyl sulfoxide. The method can obtain multiple polyploid new germplasms through one treatment under the use of a small amount of inducers. The obtained polyploid polyps can not only rapidly proliferate new individuals with polyploid characteristics through asexual reproduction, but also obtain jellyfish bodies with polyploid characteristics, such as fast growth speed, long life cycle, beautiful shape and large individual. Meanwhile, the method has the characteristics of simple operation and low cost. Therefore, the induction method has great application value and broad application prospect.

Description

Technical Field

[0001] This invention relates to the field of marine ornamental animal breeding technology, and more specifically, to a method for inducing polyploidy in jellyfish and its application. Background Technology

[0002] Jellyfish are a collective term for various animals including those in the phylum Cnidaria and phylum Ctenophora. They are important planktonic organisms in aquatic environments and are also important marine economic and ornamental animals in my country. Their life cycle includes the sexual generation of the medusa stage and the asexual generation of the polyp stage. Due to the short life cycle of medusa and the difficulty of sexual reproduction, artificial breeding has long relied on wild resources, making the research and promotion of superior varieties of jellyfish of great significance.

[0003] Polyploidy refers to organisms containing three or more sets of chromosomes. It is an important mechanism for speciation, genome evolution, and the maintenance of biodiversity, and is also a classic strategy for de novo domestication. Breeding polyploids to enhance target traits is widely used in plant breeding. However, compared to plant breeding, breeding polyploids in animals presents challenges because most animals reproduce sexually.

[0004] Among the few reported polyploids of aquatic economic animals, their polyploid offspring exhibit advantages such as larger size and faster growth compared to ordinary diploids. Therefore, in the process of aquatic organism breeding and production, polyploid breeding technology has become an important way to obtain new varieties of aquatic organisms. However, while there is more research on polyploids of aquatic organisms used to improve the germplasm resources of aquatic economic organisms, improve energy balance, and enhance the quality and yield of aquatic products, polyploid breeding of ornamental aquatic animals is rarely reported.

[0005] In view of this, the present invention is proposed. Summary of the Invention

[0006] The purpose of this invention is to provide a method for inducing polyploid jellyfish and its application. The induction method of this invention can obtain multiple new polyploid germplasm in a single treatment with a small amount of inducer. The polyploid polyps can not only rapidly proliferate through asexual reproduction while fully retaining polyploid characteristics, but also produce jellyfish with polyploid traits such as fast growth rate, long life cycle, beautiful morphology, and large size. Therefore, the induction method of this invention lays an experimental foundation for the application of polyploid induction in jellyfish.

[0007] This invention is implemented as follows: In a first aspect, the present invention provides a method for inducing polyploidy in jellyfish, comprising: induction culture and screening identification; wherein, the induction culture involves culturing jellyfish in the polyp stage in seawater containing colchicine and dimethyl sulfoxide; and the screening identification includes morphological screening, chromosome identification, and flow cytometry ploidy analysis. The mass-volume concentration ratio of colchicine in seawater is 0.01%-0.05%, and the volume concentration of dimethyl sulfoxide is 0.1%-0.5%.

[0008] Secondly, the present invention provides jellyfish polyploids obtained by the above-described induction method.

[0009] Thirdly, the present invention provides the application of the above-mentioned induction method in polyploid breeding of jellyfish.

[0010] The present invention has the following beneficial effects: (1) This invention establishes for the first time a method for inducing polyploid jellyfish by using the polyp stage of jellyfish as the induction target and treating them with a combination of colchicine and dimethyl sulfoxide. The method uses a small amount of mutagen, has low toxicity, high induction rate, and short induction time, and can quickly and efficiently obtain tetraploid polyps.

[0011] (2) The tetraploid polyps induced by the method of the present invention are in the asexual reproduction stage, so they can reproduce rapidly and maintain the stable inheritance of excellent traits. At the same time, their medusa bodies have the characteristics of long life cycle, beautiful shape, large size and easy to raise.

[0012] (3) The induction method of the present invention can be applied to tetraploid breeding of jellyfish. This method can avoid common difficulties in animals such as haploid sterility and chromosome ploidy differentiation in sexually reproduced offspring, and avoid the problem of rebreeding each generation, thereby improving the utilization rate of high-quality germplasm resources and reducing induction costs. Therefore, the induction method of the present invention has significant application value and broad prospects for promotion and application. Attached Figure Description

[0013] To more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings used in the embodiments will be briefly introduced below. It should be understood that the following drawings only show some embodiments of the present invention and should not be regarded as a limitation on the scope. For those skilled in the art, other related drawings can be obtained based on these drawings without creative effort.

[0014] Figure 1 The morphological identification results of the polyps obtained in Example 1; Figure 2 The chromosome counting results of the polyps obtained in Example 1; Figure 3 The image shows the relative DNA content of polyp somatic cells and nematodes after treatment with different concentrations of inducing agent solutions in Experiment Example 1. From left to right, the concentrations are colchicine volume concentrations of 0%, 0.01%, and 0.02%. Figure 4 This is a staining diagram of the two main types of cells in a jellyfish. Detailed Implementation

[0015] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer shall apply. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased commercially.

[0016] First, the present invention provides a method for inducing polyploid jellyfish, which mainly consists of two processes: induction culture and screening identification. The induction culture uses jellyfish in the polyp stage as material and places them in seawater containing an inducing agent for culture. The screening identification involves morphological screening, chromosome counting, and flow cytometry ploidy analysis of the induced polyps to obtain polyploid jellyfish.

[0017] Scyphozoa exhibit a typical alternation of generations life cycle, including a polyp stage (asexual fixation) and a medusa stage (sexual planktonic life). The complete cycle of alternation of generations is as follows: Fertilized egg - develops into planktonic larva ( Planula ) - Attachment of planktonic larvae - Metamorphosis into polyps ( Polyp (Asexual generation) - Polyps reproduce through transverse fission - releasing disc-shaped larvae ( Ephyra ) - Disc-shaped larvae grow - mature into jellyfish ( Medusa (Sexual generation) - jellyfish lays eggs / sperm - after fertilization, it becomes a zygote, completing the cycle.

[0018] The present invention selects the polyp as the induction target because jellyfish at this stage can rapidly proliferate through asexual reproduction, fully retaining polyploid characteristics, and can also avoid the difficulties common in animals such as haploid sterility and chromosome ploidy differentiation in sexually reproduced offspring. This overcomes the problem of rebreeding each generation, thereby improving the utilization rate of high-quality germplasm resources and reducing induction costs.

[0019] Specifically, the above-mentioned induction method includes the following steps: S1. Prepare a 20-fold inducer solution by mixing colchicine with dimethyl sulfoxide and purified water.

[0020] In the induction method of the present invention, the inducing agent is composed of colchicine and dimethyl sulfoxide.

[0021] Colchicine is the most widely used chemical mutagen, capable of inhibiting mitosis, disrupting the spindle apparatus, arresting chromosomes in metaphase, and causing chromosome doubling. However, it is a highly toxic substance, and the aforementioned effects are fatal to most animal cells. During their research, the inventors discovered that polyps have developed remarkable resilience through long-term evolution, and their asexual reproduction characteristics, similar to plants, create conditions for polyploid induction. Therefore, this invention induces polyps with colchicine, causing the polyps to bind together tubulin within the microtubules during mitosis in the asexually reproduced new individuals, preventing microtubule polymerization, thereby inhibiting spindle formation, preventing mitosis caused by changes in the cell scaffold, and ultimately leading to chromosome doubling. It is precisely because of the asexual reproduction characteristics of polyps and their remarkable resilience to colchicine that the goal of inducing diploid jellyfish into tetraploid jellyfish can be achieved.

[0022] In order to increase the solubility of colchicine, this invention adds dimethyl sulfoxide to achieve a colchicine concentration that can be achieved by preparing a 20-fold induction solution using purified water. This avoids the adverse effects of excessive fresh water on polyp rearing. At the same time, the combined use of colchicine and dimethyl sulfoxide can effectively enhance the mutagenic effect of the mutagen.

[0023] S2. Add the inducer solution prepared in S1 to seawater, and place Atlantic golden jellyfish in the polyp stage in the seawater for culture as the first generation of polyps; subculture the first generation of polyps until the second generation of polyps is obtained.

[0024] The concentration of the inducing agent solution and the treatment time significantly affect the polyploid induction effect. Higher concentrations of colchicine and dimethyl sulfoxide and treatment time can improve the induction efficiency of polyploids, but excessively high concentrations can lead to developmental arrest and organ malformations, increasing the mortality rate of the tested materials. Therefore, in actual research, appropriate inducing agent concentration and treatment time are important factors affecting the success of polyploidization. During the research in the induction system of this invention, it was found that when the concentration of dimethyl sulfoxide remained constant at 0.1%-0.5%, and the concentration of colchicine in seawater was ≤0.01%, no polyploids were produced in the newly bred polyps, which may be related to the high tolerance and permeability of the polyps. When the concentration of colchicine in seawater was >0.05%, the Atlantic golden jellyfish polyps quickly shrank, turned white, and disappeared due to the influence of multiple factors such as the concentration of colchicine dissolved in fresh water and the concentration of the inducer, resulting in a high mortality rate. When the concentration of colchicine in seawater was ≤0.05% and 0.01% < colchicine, the newly bred polyps from asexual reproduction were more likely to acquire polyploid traits, and no obvious chimeras or other ploid chromosomes were produced.

[0025] Based on this, the present invention selects the concentration of colchicine in seawater as 0.01%-0.05%, preferably 0.02%, and under this condition, the suitable volume concentration of dimethyl sulfoxide is 0.1%-0.5%.

[0026] Second-generation hydras are obtained in S2, characterized by the emergence of small buds from the foot sacs, buds, or stolons of the first-generation hydras. These buds gradually develop into second-generation hydras. The first-generation hydras are diploid, and the second-generation hydras obtained by culturing them in an inducer are tetraploid hydras.

[0027] In this invention, the second-generation polyps are derived from the asexual differentiation of cells from a certain part of the ectoderm of the first-generation polyps, thus avoiding the generation of chimeras and mixed ploids, stabilizing the genetic traits of the second-generation polyps, and reducing the incidence of malformations in dividing jellyfish.

[0028] Throughout the induction process, the temperature, salinity, and inducer concentration of the induction system remain constant. Ideally, the temperature of the induction system is maintained at 20℃-24℃, the salinity of the seawater is 20‰-32‰, and the entire induction process takes 20-30 days. Furthermore, the induction culture environment is sealed to minimize water evaporation.

[0029] To meet the needs of normal growth and reproduction of the polyps during the induction process, it is necessary to feed them Artemia nauplii every 5 days and change the water once.

[0030] During the research, the present invention induced different jellyfish and found that the induction effect of the above induction method on different jellyfish was different. Among them, the jellyfish species that can be induced to polyploidy include the Atlantic golden jellyfish, as well as some jellyfish species that can be cultured in low salinity, such as the leaf jellyfish, moon jellyfish, and all scyphozoa. Therefore, the induction method provided by the present invention can be extended to the polyploid induction research of related jellyfish.

[0031] S3. Select polyps with significantly increased size and polyps capable of transversely splitting jellyfish with excellent traits from the second-generation polyps. Then perform chromosome counting and flow cytometry ploidy analysis to screen out tetraploid polyps.

[0032] Identifying the polyploidization effect of induced polyps is a crucial step in the entire process. Morphological identification is the simplest and quickest method for identifying polyploids, and can be used for preliminary screening of mutant organisms, greatly reducing workload. However, conclusions obtained through morphological identification require further analysis using chromosome counting or DNA content determination for greater accuracy. Because the chromosomes of jellyfish in this invention are small and difficult to observe clearly under an optical microscope, and the differences in cell numbers are too large, statistical results may be inaccurate. Therefore, this invention combines chromosome counting with flow cytometry ploidy analysis. By using flow cytometry to detect newly bred polyps, polyploids in mutant individuals can be accurately identified, facilitating subsequent jellyfish breeding and selection of superior traits.

[0033] Using the above induction methods, multiple new polyploid germplasm can be obtained in a single treatment with a relatively small amount of inducer. These polyploids can not only rapidly proliferate through asexual reproduction while fully retaining polyploid characteristics, but also produce jellyfish with polyploid traits such as fast growth rate, long life cycle, beautiful morphology, and large size, which has high application prospects.

[0034] The features and performance of the present invention will be further described in detail below with reference to embodiments.

[0035] Example 1 This embodiment describes a method for inducing polyploidy in jellyfish, and the specific steps are as follows: (1) Prepare a 20-fold inducer solution by mixing colchicine with dimethyl sulfoxide and purified water.

[0036] (2) Add the inducer solution prepared in (1) to seawater to prepare an induction system with colchicine concentration of 0.02% and dimethyl sulfoxide concentration of 0.4% in seawater. Place Atlantic golden jellyfish in the polyp stage in the induction system and culture for 30 days. During this period, feed Artemia nauplii every 5 days and change the water once.

[0037] The cultivation conditions were: temperature maintained at 20℃-24℃, seawater salinity at 25‰, and the induction culture environment was sealed.

[0038] Polyps with significantly increased size and those capable of transversely splitting jellyfish with excellent traits were screened from the second-generation polyps. Then, they were counted using a modified hypotonic method and analyzed for ploidy by flow cytometry to screen out tetraploid polyps.

[0039] The modified hypotonic method was based on the karyotype study of silver pomfret chromosomes by Zhou Jianguang et al., and the specific operation method is as follows: (1) Add 4 ml of seawater with a salinity of 32‰ to 1 ml of 0.1% colchicine solution to prepare a 0.02% colchicine solution. Place the test material in the solution and culture it normally for 24 hours.

[0040] (2) To prevent interference from nematocysts, the tentacles of the polyps were cut off in advance. The tissue was dissociated with 50% glacial acetic acid, gently blown with a pipette, and allowed to stand at 37°C for 1 hour. The centrifuge tube containing the experimental material was centrifuged at 12000 r / min for 2 min, and then the supernatant was gently aspirated, leaving about 0.5 ml of cell pellet.

[0041] (3) Fix the cells with freshly prepared Carnoy's fixative for 1.5 h (0℃~-4℃). To ensure the concentration of the fixative, change the fixative twice during the period. Add 0.075 mol / L KCl solution as a hypotonic solution, blow the cells apart with a pipette, and after hot drop, air drying, Giemsa staining, and microscopic examination, select well dispersed metaphase cells for photomicrography.

[0042] Flow cytometry ploidy analysis: Jindi Future Biotechnology (Beijing) Co., Ltd. used the Sysmex Partec CyFlow Space flow cytometer to perform ploidy analysis of polyps. Specific operating procedures are as follows: Select 0.2g of newly bred, robust polyps and place them in a culture dish. Add 500ul of nuclear lysis buffer from the CyStain UV Precise P kit around the sample and use a sharp blade to chop them up to fully extract the intact nuclei. Filter the liquid in the culture dish through a 50um celltrics filter into a sample tube. Add 2000ul of DAPI fluorescent staining solution from the CyStain UV Precise P kit to the sample tube and stain in the dark for 2 minutes. Perform ploidy detection using a Sysmex Partec CyFlowSpace flow cytometer.

[0043] Figure 1 This is a morphological identification diagram. Figure 2 The result of chromosome counting. Figure 3 Flow cytometry images of different concentrations of colchicine solution (from left to right: 0%, 0.01%, 0.02%). Figure 4 These are the two main types of cells in a jellyfish.

[0044] Figure 1 These are polyps of the Atlantic golden jellyfish raised under the same conditions. The fertile tetraploid polyps exhibit the characteristics of a new tetraploid germplasm, such as large size, strong vitality of the dividing medulla, and ease of rearing. Their medulla bodies have polyploid traits such as rapid growth, long life cycle, beautiful morphology, and increased size.

[0045] Figure 2 The results are from chromosome staining of large polyps. A large number of well-dispersed metaphase cells were obtained from the experimental material, approximately twice the number of chromosomes in the polyps before treatment.

[0046] Flow cytometry detection Figure 3 The left image shows that the fluorescence intensity emitted by the stained bases in the free cell nucleus has two peaks. The first main peak is located near the fluorescence intensity, i.e., DNA content of 19, indicating the detection of 77 fluorescently labeled cells. The second main peak is located near the fluorescence intensity of 55, indicating the detection of 238 cells. This is consistent with the finding that the polyp mainly contains two types of cells of different sizes: nematocysts and somatic cells (see [link to image]). Figure 4 ). Figure 3 The intermediate image results and Figure 3 Similar to the left image, both show two peaks in fluorescence intensity around 19 and 55, indicating that both are diploid. Figure 3 In the right figure, the two peaks are located near fluorescence intensities of 38 and 105, respectively, indicating that both stinging cells and somatic cells double under the influence of the drug.

[0047] Example 2 The difference from Example 1 is that the induced object in this experiment is the polyp of Andorra jellyfish.

[0048] Example 3 The difference from Example 1 is that the inducer in this experiment is the polyp of the moon jellyfish.

[0049] Comparative Example 1 The difference from Example 1 is that colchicine solution was not added in this experiment.

[0050] Comparative Example 2 The difference from Example 1 is that the concentration of colchicine solution in this experiment is 0.01%.

[0051] Comparative Example 3 The difference from Example 1 is that the concentration of colchicine solution in this experiment is 0.05%.

[0052] Experimental Example 1 The polyps obtained in Example 1 and Comparative Examples 1-3 were screened and identified, and the results of flow cytometry ploidy analysis are as follows: Figure 3 As shown.

[0053] The results show that when Atlantic golden jellyfish polyps were cultured in a 0.01% colchicine solution for 30 days, they produced more individuals through asexual reproduction, with a survival rate of 126.7%. Each polyp produced an average of 4.50 discoids through complex transverse fission, which is close to the survival rate of 132% and the average number of discoids produced per polyp in natural seawater (4.67). Long-term culture with 0.01% colchicine did not change the ploidy of the polyps. Flow cytometry analysis showed that the DNA content was similar to that of naturally cultured polyps, with the two main peaks located around fluorescence intensities of 19 and 55, respectively.

[0054] Under 0.02% colchicine conditions, the survival rate of polyps was 40%, but some polyps produced new polyps through asexual reproduction. Flow cytometry results showed that the two main peaks of nematocyst and somatic cell DNA content in newly produced polyps were located near fluorescence intensities of 38 and 105, respectively, indicating that almost all were tetraploid. Under 0.02% colchicine conditions, each polyp produced 2.27 discoids, mainly due to the large number of polyps undergoing complex transverse fission in the initial stage of the experiment, possibly related to the drastic change in the rearing environment. In this example, some tetraploid polyps underwent transverse fission, producing highly vigorous and rapidly growing discoids.

[0055] The study also compared the induction effects of colchicine concentrations exceeding 0.01%-0.05%. When the concentration of colchicine in seawater was ≤0.01%, no polyploids were produced in the newly bred polyps. When the concentration of colchicine in seawater was ≥0.05%, the Atlantic golden jellyfish polyps quickly shrank, turned white, and disappeared due to the influence of multiple factors such as the concentration of dissolved colchicine in freshwater and the concentration of the inducer, resulting in a high mortality rate.

[0056] The above results indicate that the polyploid hydra obtained by this invention can reproduce asexually and split transversely to produce disc-shaped bodies. The offspring can grow normally, and the asexually reproduced hydra individuals can maintain their polyploid traits. The polyploid hydra obtained has no mechanical damage, the first generation survival rate is over 60%, the second generation survival rate is 100%, the second generation shows tetraploids, and the polyploid induction rate is 100%.

[0057] Experimental Example 2 The polyps obtained in Example 1 and Example 2 were compared, and the results showed that the polyps of *Andrographis paniculata* reproduced asexually through budding. When cultured in a seawater induction system with colchicine concentration of 0.02% and dimethyl sulfoxide concentration of 0.4% for 30 days, and fed with Artemia nauplii every 5 days and with water changed once, tetraploids were produced.

[0058] Experimental Example 3 The polyps obtained in Example 1 and Example 3 were compared. The results showed that the polyps of the moon jellyfish reproduced asexually through various means such as stolon reproduction, foot sac reproduction, and budding reproduction. When cultured at 20-25℃ in a seawater induction system with a colchicine concentration of 0.02% and a dimethyl sulfoxide concentration of 0.4% for 30 days, and fed with Artemia nauplii every 5 days and with water changed once, tetraploids could be produced.

[0059] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A method for inducing polyploidy in jellyfish, characterized in that, include: Induction culture and screening identification; The induction culture involves culturing jellyfish in the polyp stage in seawater containing colchicine and dimethyl sulfoxide. The screening and identification process includes morphological screening, chromosome counting, and flow cytometry ploidy analysis. The colchicine concentration in seawater is 0.01%-0.05% by volume, and the dimethyl sulfoxide concentration is 0.1%-0.5% by volume. The induction culture includes: placing the polyp stage of the jellyfish in seawater containing colchicine and dimethyl sulfoxide to raise it as the first generation of polyps; the first generation of polyps is then cultured in a subculture to produce a new generation of polyps through asexual reproduction in seawater containing colchicine and dimethyl sulfoxide, which are then raised as the second generation of polyps; and the second generation of polyps is then split horizontally to produce jellyfish bodies. The conditions for the induction culture are: temperature of 20℃-24℃, time of 20-40 days, salinity of seawater of 20‰-32‰, and the environment for the induction culture is a sealed state; The screening and identification process involves: selecting polyps with significantly increased size and polyps capable of transverse splitting to produce jellyfish with superior traits from the obtained second-generation polyps, and then performing chromosome counting and flow cytometry ploidy analysis to screen out tetraploid polyps.

2. The induction method according to claim 1, characterized in that, During the induction culture process, Artemia nauplii were fed every 5 days and the water was changed once.

3. The induction method according to claim 2, characterized in that, The colchicine solution used in the induction culture is a 20-fold solution prepared by dissolving dimethyl sulfoxide and purified water, and the mass-volume concentration of colchicine in seawater is 0.02%.

4. The induction method according to claim 1, characterized in that, The jellyfish mentioned are scyphozoans, including the Atlantic golden jellyfish, the moon jellyfish, and sea jellyfish.

5. The application of the induction method as described in any one of claims 1-4 in polyploid breeding of jellyfish.

6. The application according to claim 5, characterized in that, The polyploid breed is a tetraploid breed.

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