Method for improving plant polyploidy induction rate based on leaf bud primordium cell cycle synchronization and obtaining polyploidy regenerated plant

Abstract

The invention discloses a method for improving plant polyploidy induction rate and obtaining polyploidy based on leaf bud primordium cell cycle synchronization. The method comprises the following steps: carrying out pre-differentiation culture on an explant induction material; performing cell cycle synchronization excitation treatment on the leaf bud primordium cells obtained by the pre-differentiation treatment; and performing chromosome doubling treatment on the explant induction material subjected to the cell cycle synchronization excitation treatment. According to the method disclosed by the invention, an EdU staining labeling method and a flow cytometry are used for accurately judging the time for carrying out cell cycle synchronization excitation treatment on leaf bud primordium cells of an induction material, and carrying out synchronization excitation and doubling induction on the induction material in time; the dosage of a cell cycle inhibitor for synchronous excitation treatment is accurate, the treatment opportunity is accurate, the treatment duration is proper, and the polyploidy inductivity of the plant is remarkably improved; the obtained polyploidy plant is good in stability, and the method is high in repeatability.

Description

Technical Field

[0001] The invention relates to a method for inducing plant polyploid, in particular to a method for improving the induction rate of plant polyploid, and belongs to the field of plant biotechnology. Background Art

[0002] One of the current ways to obtain polyploid plants is to use physical and chemical means to induce somatic cell chromosome doubling. The main principles include affecting the normal function of the spindle in the metaphase of cell division in the leaf bud primordium, or affecting the cytoplasmic movement and cell plate formation in the late phase of division. In short, the key cell cycle for inducing polyploid formation is the division phase (M phase). As we all know, the M phase is the shortest period in the entire cell cycle, and under normal circumstances, the number of M phase cells in plant induction materials is very low. Therefore, the most important step in conducting plant polyploid induction research is to find the key node where the number of M phase cells in the doubling material accounts for the largest proportion. Traditional research strategies usually first construct a series of real-time discrimination indicators based on the morphological changes of the induced materials, divide the morphological changes into different stages based on the real-time discrimination indicators, and conduct a large number of cytological observations on these materials at different stages by combining paraffin sections and other technical means, and link the discrimination indicators with the development process of the cell cycle to guide subsequent doubling studies (Congping Xu, Ying Zhang, Zeng Huang, Pengqiang Yao, Yun Li, Xiangyang Kang. Impact of the Leaf Cut Callus Development Stages of Populus on the Tetraploid Production Rate by Colchicine Treatment. [J] Journal of Plant Growth Regulation, 2017). However, many studies on ploidy breeding have shown that even if the optimal induction period is determined, the proportion of cells that can effectively double during this period is not high. This is due to the asynchrony of cell cycle development, which reduces the induction efficiency of polyploids (Kang Xiangyang, Wang Jun. Research on Polyploid Induction Technology of Poplars, Science Press, 2010).

[0003] A common strategy for stimulating cell cycle synchronization is to perturb the cells by physical, chemical or biological means (a process referred to as capture) and then remove the source of perturbation (i.e. release). The capture process will cause certain damage to the cell's DNA, organelles, etc., which will then trigger the cell's inspection and repair mechanism, causing some cells to enter a state of cell cycle arrest (Campo D, Samaniego R, Giménez-Abián JF, et al. G2 checkpoint targets latereplicating DNA. [J] Biology of the Cell, 2003; Fischer JA, Caradonna S. Analysis of Nuclear Uracil–DNA Glycosylase (nUDG) Turnover During the Cell Cycle. In. Cell Cycle Synchronization. Springer, 2011). When the interference source is removed, a large number of cells that are arrested in the same period will advance at the same time, which can achieve the effect of increasing the proportion of the target cell cycle in a short period of time (Senovilla L, Demont Y, Humeau J, Bloy N, Kroemer G. Cell Cycle Synchronization. UK: Humana Press, 2017).Studies have shown that the synchronization effect obtained by capturing the cell cycle using physical methods is not accurate. It will affect and arrest the cell cycle at different stages. For example, UV-B irradiation can cause the cell cycle to arrest in G1, S or G2 / M phases (Athar M, Kim AL, Ahmad N, Mukhtar H, Gautier J, Bickers DR. Mechanism of ultraviolet B-induced cell cycle arrest in G2 / M phase in immortalized skinkeratinocytes with defective p53. [J] Biochemical and biophysical research communications, 2000). Therefore, after the interference is removed, the cycles of most cells are still out of sync (Liu B. A Preliminary Investigation on the Synchronization of HepatocellularullarCarcinoma Cells and its Adhesive Properties with extracellular Matrix. [M] Chongqing University, 2002). Moreover, physical and biological methods (such as starvation method) are prone to cause irreversible damage to cells, which will affect the doubling effect after cell cycle synchronization. The application of cell cycle inhibitors can accurately capture the target cell cycle, causing most cells to stagnate relatively neatly at the same stage. In addition, the damage caused by cell cycle inhibitors is usually reversible under appropriate treatment (Khammanit R, Chantakru S, Kitiyanant Y, Saikhun J. Effect of serum starvation and chemical inhibitors on cell cycle synchronization of canine dermal fibroblasts. [J] Theriogenology, 2008), which ensures the synchronization effect and is also conducive to subsequent doubling induction.

[0004] There are many cell cycle inhibitors that can increase the number of cells in the M phase. In the study of plant cell cycle synchronization induction, aphidicolin (APH), bleomycin (Zeocin), and pyrimidine nucleoside analogs are all commonly used cell cycle inhibitors. APH is an effective inhibitor of cell DNA synthesis. Cells treated with it will be enriched in large quantities in the S phase and enter the M phase relatively synchronously after the drug is released (Weingartner M, Criqui MC, Meészaéros T, et al. Expression of a nondegradable cyclin B1 affects plant development and leads to endomitosis by inhibiting the formation of a phragmoplast. [J] The Plant Cell, 2004). Bleomycin also blocks cells in the S phase, but to a large extent, its impact on cells is more serious. In the study of endoreduplication in Arabidopsis, it was found that bleomycin is more likely to induce the production of "large cells" in the root tips of Arabidopsis than cisplatin and radiation, because bleomycin can seriously damage the single and double strands of DNA, making the damaged cells unable to function normally and enter the state of endoreduplication to form "large cells". The number of chromosome sets in such "large cells" can even reach 36C (Adachi S, Minamisawa K, Okushima Y, et al. Programmed induction of endoreduplication by DNA double-strand breaks in Arabidopsis. [J] Proceedings of the National Academy of Sciences, 2011). Some studies have compared 5-aminouracil (5-AU) with other cell cycle inhibitors, including APH. The results show that when the percentage of aberrant mitosis is low, the inhibitor with the highest mitotic index is 5-AU ( Winnicki K,Polit JT,Bernasińska- J, Maszewski J. 5-Aminouracil and other inhibitors of DNA replication induce biphasic interphase–mitotic cells in apical root meristems of Allium cepa. [J] Plant Cell Reports, 2020), indicating that 5-AU causes less damage to cell materials under the premise of similar cell cycle synchronization induction effect.

[0005] Since cell cycle inhibitors mainly work in a specific cell cycle, the use of cell cycle inhibitors to achieve cell cycle synchronization first requires accurate identification of the developmental process of the cell cycle in plant materials, which is crucial for determining the synchronization stimulation period and identifying the synchronization effect. It is helpful to determine the appropriate synchronization treatment node and treatment duration. Otherwise, in a blind situation, the cell cycle inhibitor will not work in the correct cell cycle, and it is also impossible to determine how long the treatment will take to completely cover the effective action cycle, which will ultimately fail to achieve a good cell cycle synchronization effect.

[0006] In this regard, EdU staining and labeling technology and flow cytometry have provided support for the development of this technical system (Xia Y, Cao Y, Ren Y, et al. Effect of a suitable treatment period on the genetic transformation efficiency of the plant leaf disc method. [J] Plant Methods, 2023). The present invention specifically labels S phase cells through EdU staining and labeling, judges the changes in S phase cells in plant materials by the number and density of fluorescence, and clearly judges the proportion of each cell cycle in different developmental periods based on flow cytometry. Combining the two, the occurrence and development process of the cell cycle in plant materials can be clearly judged, and then the method of applying 5-AU can be determined.

[0007] The present invention uses the cell cycle inhibitor 5-AU to develop a method for improving the efficiency of plant polyploid induction based on plant cell cycle synchronization. The method of the present invention is of great significance for enriching the plant polyploid induction theory and constructing a more efficient forest polyploid induction system. Summary of the invention

[0008] The purpose of the present invention is to solve the technical problems of low induction rate and instability in the existing plant somatic cell chromosome doubling process, and provide a method for improving the induction efficiency of plant polyploidy based on the synchronization of leaf bud primordium cell cycle. The method of the present invention accurately judges the timing of the leaf bud primordium cells in the plant material in the S cycle according to the specific development process of the leaf bud primordium cells in the differentiation and culture process of the induction material, and applies a cell cycle inhibitor to accurately capture the target lower cycle, so that a large number of target cells in the treatment interphase are relatively neatly stagnant at the same stage (i.e., the S cycle stage of the interphase), control the synchronization excitation and chromosome doubling period, thereby ensuring the synchronization of the leaf bud primordium cell cycle, and significantly improves the induction rate and yield of plant polyploids after release, as well as the stability of high induction efficiency.

[0009] To achieve the purpose of the present invention, the following technical solutions are used:

[0010] A method for improving plant polyploid induction efficiency based on leaf bud primordium cell cycle synchronization comprises: firstly, during differentiation culture of sterile plant explant materials, using cell cycle inhibitors to perform cell development cycle synchronization stimulation treatment on leaf bud primordium cells; and then using chromosome doubling inducers to perform induction treatment on the explants after the cell cycle synchronization stimulation treatment.

[0011] Wherein, the explant material is a leaf or stem segment of an in vitro cultured plant, preferably a leaf.

[0012] In particular, the cell cycle inhibitor is 5-aminouracil (5-AU), aphidicolin (APH) or bleomycin (Zeocin); the chromosome doubling inducer is colchicine, oryzalin or gibberellin.

[0013] In particular, it also includes first identifying the occurrence and development process of leaf bud primordium cells of differentiated explant materials, and when the leaf bud primordium cells of differentiated explants enter the S phase and the proportion of S phase cells begins to increase, that is, when they are in the S phase cell growth period, the explant material is subjected to cell cycle synchronization stimulation treatment to enrich the S phase cells.

[0014] A method for improving the efficiency of plant polyploid induction based on synchronization of leaf bud primordium cell cycles comprises the following steps performed in order:

[0015] 1) Inoculating the explant material of the sterile plant into the differentiation medium for pre-differentiation culture;

[0016] 2) To identify the developmental process of leaf bud primordium cells obtained by pre-differentiation culture of explant materials;

[0017] 3) applying a cell cycle inhibitor according to the developmental process of the leaf bud primordium cells, and performing a cell development cycle synchronization stimulation treatment on the leaf bud primordium cells of the explant material, wherein the cell development cycle synchronization stimulation treatment is to use the cell cycle inhibitor to induce the leaf bud primordium cells of the explant material to stagnate in the S phase;

[0018] 4) sequentially removing the drug from the explant material after the cell development cycle synchronization stimulation treatment and performing recovery culture;

[0019] 5) Applying a chromosome doubling inducing agent to the explant material after recovery culture, and performing the chromosome doubling inducing treatment under light-proof conditions.

[0020] Wherein, the explant material in step 1) is a leaf or stem segment of an in vitro cultured plant, preferably a leaf.

[0021] In particular, the explant material is a leaf or stem tip of Populus argentatus 84K, preferably a leaf. In addition to Populus argentatus 84K, Populus tomentosa, Populus simonii, etc. are all suitable for the present invention.

[0022] In particular, the explant material is the functional leaves of the silver gland poplar 84K tissue culture seedlings that have been aseptically cultured for 4-5 weeks, preferably the leaves at the 4th to 5th leaf positions, counted from the top leaves.

[0023] Among them, the pre-differentiation culture treatment in step 1) is to inoculate the silver gland poplar 84K sterile plant explant material into the differentiation culture medium for differentiation culture.

[0024] Particularly, the explant material is pretreated as follows and then inoculated into a differentiation medium, a wound is cut perpendicular to the main vein of the leaf, the main vein is cut off, and the leaf is not cut off.

[0025] In particular, the differentiation medium is: MS basic medium + NAA 0.005-0.05 mg / L + 6-BA 0.1-0.5 mg / L + agar 6 g / L + sucrose 30 g / L, preferably MS basic medium + NAA 0.05 mg / L + 6-BA 0.5 mg / L + agar 6 g / L + sucrose 30 g / L.

[0026] Particularly, the pre-differentiation culture conditions are: temperature of 25±2°C; light intensity of 2000lx (usually 1500-2500lx); light cycle of 16h light / 8h dark (usually 10-16h light / 8-14h dark).

[0027] In particular, in step 2), the occurrence and development process of the leaf bud primordium cells obtained by the pre-differentiation culture of the explant material is judged, and after confirming that the leaf bud primordium cells obtained by the pre-differentiation culture of the explant material have entered the S phase, the cell development cycle synchronization stimulation treatment is then performed.

[0028] In particular, the method for distinguishing the occurrence and development process of leaf bud primordium cells in step 2) includes: using EdU fluorescence labeling method to detect the cell proliferation dynamics of explant materials at different stages of pre-differentiation culture, and observing the changes in EdU fluorescence signals of leaf bud primordium cells entering the S phase in callus tissue through fluorescence microscopy; or using flow cytometry to detect callus tissue at different stages and analyze the distribution characteristics of the cell cycle of explant materials.

[0029] Combining the two methods (EdU fluorescence labeling and flow cytometry), the occurrence and development stages of leaf bud primordium cells in explant materials were accurately determined, thus providing a basis for subsequent experiments to implement cell cycle synchronization stimulation and chromosome doubling treatment in the optimal time window. Determine the treatment period for explant materials to undergo cell development cycle synchronization stimulation treatment.

[0030] In particular, in the EdU staining test, when it is observed that the cells emit green fluorescence, which means that the cells have developed to the S phase and the number of cells in the S phase has increased (that is, the leaf bud primordium cells in the S phase are in the growth phase), the explant material is subjected to a cell development cycle synchronization excitation treatment to enrich the S phase cells; the flow cytometry test is to analyze the development cycle proportion of leaf bud primordium cells of callus tissue by flow cytometry. When it is observed that the proportion of S phase cells begins to increase, that is, when the cells are in the growth phase of the S phase, the explant material is subjected to a cell development cycle synchronization excitation treatment to enrich the S phase cells, so that a large number of them are in the S phase.

[0031] In particular, according to the development process of leaf bud primordium cells, cell cycle inhibitors are used to perform cell synchronization stimulation treatment, wherein the cell synchronization stimulation is to use cell cycle inhibitors to synchronize the cell cycle during cell development. In the present invention, cell cycle inhibitors are used to induce leaf bud primordium cells of explant materials to stagnate in the S phase.

[0032] When EdU staining was performed, partial green fluorescence was observed in the plant induction material (no large amount of green fluorescence appeared), followed by a large amount of dense green fluorescence, or when flow cytometry was used to detect the incision site of the induction material, it was detected that the proportion of the S cell cycle began to increase. This period of time was defined as the S phase growth period.

[0033] Among them, the cell cycle synchronization stimulation in step 3) includes using cell cycle inhibitors to perform cell development cycle synchronization stimulation treatment on the explant material determined to be in the S phase growth period of leaf bud primordium cells, that is, the explant material when the proportion of leaf bud primordium cells determined to be in the S phase increases is treated with cell cycle inhibitors.

[0034] In particular, the cell development cycle synchronization stimulation treatment in step 3) is: adding cell cycle inhibitors to the explant material that is in the S phase growth stage of leaf bud primordium cells after pre-differentiation culture treatment and identification of the developmental process of leaf bud primordium cells, and performing cell development cycle capture treatment under light-proof conditions, that is, using cell cycle inhibitors to stimulate the S phase synchronization of leaf bud primordium cells, so that leaf bud primordium cells stagnate in the S phase and enrich S phase cells.

[0035] In particular, the cell development cycle synchronization stimulation treatment (i.e., capture treatment) described in step 3) is performed on differentiation medium under light-proof conditions by adding cell cycle inhibitors when the leaf bud primordium cells obtained by pre-differentiation culture of the explant material are in the S phase of growth.

[0036] In particular, the cell cycle inhibitor is one of 5-aminouracil (5-AU), aphidicolin (APH), and zeocin, preferably 5-AU.

[0037] Particularly, the concentration of the cell cycle inhibitor is 1-2 mM, preferably 1-1.7 mM, and more preferably 1-1.5 mM.

[0038] Particularly, the capture treatment conditions are: temperature of 25±2°C, dark culture; capture treatment duration of 12-24h, preferably 24h.

[0039] Specifically, 5-AU at a concentration of 1.5 mM was used for cell synchronization capture treatment for 24 h.

[0040] Particularly, the capture treatment is to drop a cell cycle inhibitor (preferably a 5-AU solution) onto the incision of the plant induction material in the S phase of growth, and culture in the dark.

[0041] In particular, the concentration of the cell cycle inhibitor and the duration of the synchronized stimulation treatment for the cell development cycle in step 3) are determined using the comet assay method.

[0042] The DNA damage of plant-induced materials was observed by comet assay to screen the tolerance range of explants to 5-AU; the duration of 5-AU light-proof treatment (capture treatment) was identified by EdU staining and flow cytometry, and the duration of the S phase growth period was determined. In the present invention, the light-proof treatment was 24 hours.

[0043] In particular, the drug removal treatment in step 4) is: after taking out the explant material after the capture treatment, soaking and washing it with sterile distilled water for at least 3 times (usually 3-5 times) and absorbing the water;

[0044] In particular, the recovery culture in step 4) is: inoculating the explant material after drug removal treatment into differentiation medium, and performing recovery culture treatment under light conditions for at least 20 hours, preferably 24-48 hours.

[0045] In particular, during the recovery culture process, the temperature is controlled at 25±2°C; the light intensity is 1500-2500lx, preferably 2000lx; and the light cycle is 10-16h light / 8-14h dark, preferably 16h light / 8h dark.

[0046] When the explant leaf bud primordium cells were recovered after drug release treatment, the cell cycle progress was relatively synchronized during the recovery culture process.

[0047] In particular, the chromosome doubling induction treatment in step 5) includes: after stimulating the cell cycle synchronization of the leaf bud primordium of the explant material by colchicine induction or other chromosome doubling induction methods, when leaf bud primordium cells with nuclear morphology in the G2 / M phase are observed by EdU labeling and staining; or when the cell cycle ratio is analyzed by flow cytometry and the G2 / M phase cell ratio begins to appear, the induced material is subjected to chromosome doubling treatment to obtain a higher polyploidy induction rate.

[0048] In particular, before the chromosome doubling induction treatment is carried out, the method also includes distinguishing and identifying the leaf bud primordium cells of the explant material after the recovery culture, including the following steps: performing EdU staining detection on the explant material after the recovery culture to determine the change in the number of cells in the leaf bud primordium cells of the explant material after the recovery culture entering the G2 / M phase; or performing flow cytometry detection on the explant material after the recovery culture to determine the G2 / M cell ratio of the explant material, and distinguishing the timing of the chromosome doubling induction treatment for the explant material.

[0049] The chromosome doubling induction treatment in step 5) is to add a chromosome doubling inducer to the explant material after the cell cycle synchronization stimulation treatment, and perform the induction treatment on a differentiation medium in the dark for at least 2 days, preferably 2-4 days.

[0050] In particular, the chromosome doubling induction treatment includes: using colchicine induction method or other chromosome doubling induction methods to synchronize the cell cycle of leaf bud primordium of explant material, de-drug treatment, and recovery culture, and when leaf bud primordium cells in G2 / M phase are observed by EdU labeling staining; or when the cell cycle ratio is analyzed by flow cytometry and the ratio of G2 / M phase cells begins to increase, the induced material is subjected to chromosome doubling treatment to improve the polyploidy induction rate.

[0051] During the cell synchronization and excitation treatment, 5-AU was added for synchronization and excitation treatment when edu staining and flow cytometry were used to observe the leaf bud primordium cells in the S phase; during the colchicine chromosome doubling treatment, colchicine solution was added for chromosome doubling treatment when edu staining and flow cytometry were used to observe the leaf bud primordium cells in the G2 / M phase.

[0052] Particularly, the chromosome doubling inducing agent is colchicine, oryzalin, or gibberellin, preferably colchicine.

[0053] Particularly, the chromosome doubling inducer is a colchicine solution with a concentration of 40-60 mg / L, preferably 50 mg / L.

[0054] In particular, the chromosome doubling process is performed according to the following steps:

[0055] A) Prepare a 50 mg / L sterile colchicine aqueous solution under light-proof conditions for later use;

[0056] B) dripping a sterile colchicine aqueous solution onto the inducing material treated with cell cycle synchronization stimulation, and performing a chromosome doubling induction treatment on a differentiation medium in a dark condition for 2-4 days (preferably 3 days);

[0057] C) After the chromosome doubling induction treatment, the induction material is taken out and soaked and rinsed with sterile distilled water for at least 3 times (preferably 3-5 times); after the surface moisture of the induction material is dried, it is inoculated on the differentiation medium and recovered for at least 20 hours (preferably 24-48 hours).

[0058] The colchicine solution is filtered and sterilized before use; the colchicine solution is dripped onto the incision of the plant induction material, and the dripping amount is 1±0.2 ml per incision of each leaf; and the material is cultured in the dark for 2-4 days, preferably 3 days.

[0059] Before the chromosome doubling treatment, the explant material cell cycle synchronization stimulation is judged by comparing the amount of green fluorescence of the synchronized stimulated and unstimulated induced materials by EdU staining, or / and comparing the proportion of S phase and G2 / M phase cells of the synchronized stimulated and unstimulated induced materials by flow cytometry to determine the stimulation effect of cell cycle synchronization and the growth period of G2 / M phase. Finally, the chromosome doubling induction is carried out during the growth period of G2 / M phase.

[0060] Another technical solution of the present invention is to provide a method for obtaining polyploid regenerated plants based on synchronization of leaf bud primordium cell cycles, comprising: firstly, performing polyploidy induction treatment on explant materials according to the above-mentioned method for improving plant polyploidy induction efficiency based on synchronization of leaf bud primordium cell cycles; then, performing differentiation culture on the explant materials after the polyploidy induction treatment on a differentiation medium until adventitious buds are differentiated and cultured; then, the adventitious buds are inoculated on a rooting medium for rooting culture to obtain polyploid regenerated plants.

[0061] Among them, the rooting medium is 1 / 2MS basic medium + NAA 0.01-0.03mg / L + IBA 0.03-0.05mg / L + agar 6g / L + sucrose 30g / L, preferably 1 / 2MS basic medium + NAA 0.02mg / L + IBA 0.05mg / L + agar 6g / L + sucrose 30g / L.

[0062] In particular, the differentiation culture conditions and rooting culture conditions are: the temperature is controlled at 25±2°C; the light intensity is 2000lx (usually 1500-2500lx); the photoperiod is 16h light / 8h dark (usually 10-16h light / 8-14h dark).

[0063] In particular, the method also includes identifying the ploidy of the regenerated plants by using flow cytometry and chromosome flattening observation. The chromosome flattening observation is to stain the chromosomes of the cells of the regenerated plants with Carbopol stain and observe them under a microscope.

[0064] The cell cycle synchronization is to use the cell cycle inhibitor 5-aminouracil (5-Aminouracil, 5-AU) to specifically block the leaf bud primordium cells in the S phase of the induction material, and after the S phase cells are enriched, they are released by drug removal, and a large number of S phase cells develop synchronously, so that the number of M phase cells that are easy to double chromosomes is significantly increased. Based on this technology, colchicine is used for chromosome doubling treatment, which can effectively improve the polyploid induction rate. This method combines the comet test detection and other means to quickly determine the applicable concentration range and treatment time of the cell cycle inhibitor, and also combines the EdU staining labeling method and flow cytometry and other means to accurately distinguish the S phase of the leaf bud primordium cell cycle of the induction material, ensuring that the induction material is synchronized and stimulated and doubling induced in time.

[0065] The method of improving plant polyploid induction efficiency based on cell cycle synchronization of the present invention has the following advantages:

[0066] 1. The method of the present invention can quickly determine the excitation system of plant leaf bud primordium cell synchronization and the appropriate period of chromosome doubling induction, thereby improving the efficiency and repeatability of plant polyploid induction; and the polyploid induction efficiency of plants treated by the method of the present invention is significantly improved. It has important reference significance for the establishment and optimization of polyploid induction of plants of the same type.

[0067] 2. The method of the present invention helps to quickly determine the induction system for stimulating the cell cycle synchronization of plant induction materials, avoiding the need to determine the appropriate stimulation conditions through a large number of cumbersome orthogonal experiments due to the existence of too many factors and levels.

[0068] 3. The present invention can instantly determine the cell cycle development process of plant induction materials based on multiple discrimination methods, thereby facilitating the instantaneousness, effectiveness and reliability of the operation.

[0069] That is, the present invention uses EdU staining and flow cytometry to observe and measure the occurrence and development process of leaf bud primordium cells at the incision of the induction material that has been pre-differentiated, accurately judges the development period of the leaf bud primordium, and uses cell cycle inhibitors to perform cell cycle synchronization and stimulation treatment on the induction material during the growth period of the leaf bud primordium cells from the development to the S phase of the cell cycle, thereby avoiding the blindness of the synchronization and stimulation treatment, accurately grasping the timing of the cell cycle synchronization and stimulation treatment, increasing the number of cells in the S phase and significantly increasing the number of cells in the M phase that are easy to double chromosomes;

[0070] 4. The present invention overcomes the problem that when inducing polyploid plants in the prior art, there are problems such as large fluctuations in chromosome doubling efficiency and difficulty in obtaining stable and efficient plant polyploids due to different times for differentiating leaf bud primordium cells from in vitro cultured induction materials of different plants or different genotypes of the same plant, or different times for leaf bud primordium cells to enter the DNA replication phase due to differences in culture conditions.

[0071] 5. In the method of the present invention, when most of the leaf bud primordium cells of the explant material develop to the G2 / M phase, colchicine is used to induce chromosome doubling. The timing of the chromosome doubling treatment is precisely controlled, which significantly enhances the timeliness of chromosome doubling, thereby ensuring that the polyploid induction efficiency will not be affected by differences in the culture environment, species or genotype, thus laying an important methodological foundation for improving the polyploid induction efficiency of plants and obtaining a large number of highly stable polyploids.

[0072] The method of the present invention is based on the developmental state of leaf bud primordium cells of plant induction materials. Through EdU staining and flow cytometry, the nodes of the induction material cells developing to the S and G2 / M phases of the cell cycle are determined, the timing of cell cycle synchronization stimulation and chromosome doubling treatment is accurately controlled, the efficiency of plant polyploid induction is significantly improved, and a large number of plant polyploids with high stability are obtained. BRIEF DESCRIPTION OF THE DRAWINGS

[0073] Figure 1 Pretreat the leaves before in vitro differentiation culture of Populus alba 84K.

[0074] Figure 1A These are flow cytometric images of cell development during the continuous differentiation of in vitro cultured leaves of Populus alba 84K. 1-4 are flow cytometric peak images of the leaf cuts at 24h, 48h, 72h and 96h of differentiation culture.

[0075] Figure 1B These are the EdU fluorescence-labeled images of cell development during the continuous differentiation of in vitro cultured leaves of Populus alba 84K. 1-4 are the EdU fluorescence-labeled images of the leaf cuts at 24h, 48h, 72h and 96h of differentiation culture, respectively.

[0076] Figure 1C The cell nucleus morphology labeled by EdU at different stages of cell development, where 1 represents cells in the early S phase; 2 represents cells in the late S phase; and 3 represents cells in the G2 / M phase.

[0077] Figure 2 The leaf morphology of in vitro cultured leaves of Populus alba 84K was immersed in 5-AU with concentrations of 0.5, 1, 1.5 and 2 mM for 24 hours, washed out of the solution and then placed in differentiation medium for one week.

[0078] Figure 2A Single cell electrophoresis images of explants after treatment with different concentrations of 5-AU for 12 h and 24 h, where 1-5 are treated with various concentrations (1.1, 1.3, 1.5, 1.7 and 1.9 mM) for 12 h; 6-10 are treated with various concentrations for 24 h.

[0079] Figure 2B is the DNA tail length of cells treated with different concentrations of 5-AU, the treatment concentrations are 1.1, 1.3, 1.5, 1.7 and 1.9 mM, respectively. The 5-AU treatment time of 12 h is the square dotted line, and 24 h is the circle dotted line.

[0080] Figure 3 The flow cytometric peak diagram of 84K leaves after treatment with different concentrations of 5-AU for 24 hours, de-drug treatment, and recovery culture in differentiation medium for one day, where a, b, c, and d correspond to 1.1, 1.3, 1.5, and 1.7 mM 5-AU, respectively.

[0081] Figure 3A The figure shows the cell cycle status of the group treated with 1.5 mM 5-AU at 72 h and 96 h of differentiation, where a and b are the flow cytometry peak graphs at 72 h and 96 h of differentiation, respectively.

[0082] Figure 3BThe cell cycle status of the group treated with 1.5 mM 5-AU at 72 h and 96 h of differentiation, where a and b are EdU fluorescence labeling images at 72 h and 96 h of differentiation, respectively.

[0083] Figure 4 This is the observation of chromosomes in 84K shoot apical meristem cells. a is the growing point cell group without treatment, b is the growing point cell group treated with 5-AU, c is the growing point cell group at 40h of the release period, and d is the chromosome bridge phenomenon caused by 5-AU.

[0084] Figure 4A This is a curve diagram of the dynamic changes in the cell division index (MI) in the shoot apex meristem during the capture period and the release period. During the release period, the shoot apex of 84K tissue culture seedlings was pressed and observed every 8 hours, and the MI was calculated. The observation was continued until the 56th hour of the release period.

[0085] Figure 5 This is a diagram showing the identification results of the regenerated plants of Populus argentatus 84K; a and b are flow cytometry images of the diploid 84K regenerated plants and the tetraploid 84K regenerated plants, respectively.

[0086] Figure 5A The figure shows the identification results of the regenerated plants of Populus argentatus 84K; a and b are the cell chromosome compression observations of the diploid 84K regenerated plants (2n=2x=38) and the tetraploid 84K regenerated plants (2n=4x=76), respectively. Specific implementation methods

[0088] The present invention will be further described below in conjunction with specific embodiments, and the advantages and features of the present invention will become clearer as the description proceeds. However, these embodiments are exemplary only and do not constitute any limitation to the scope of the present invention. It should be understood by those skilled in the art that the details and forms of the technical solution of the present invention may be modified or replaced without departing from the spirit and scope of the present invention, but these modifications and replacements all fall within the scope of protection of the present invention.

[0089] Example 1 Experimental Materials

[0090] 1. Plant materials

[0091] The invention uses sterile leaves and stem tips of silver gland poplar 84K tissue culture seedlings as plant induction materials (ie explants) to carry out cell cycle synchronization stimulation and chromosome doubling.

[0092] In addition to the leaves and stem tips of Populus argentatus 84K, other plant materials such as broad-leaved plants such as Populus tomentosa, Populus simonii, Eucalyptus or Acacia are also suitable for the method of the present invention.

[0093] 2. Plant growth regulators

[0094] The plant growth regulators used in the present invention are α-naphthylacetic acid (NAA), 6-benzylaminopurine (6-BA) and indole-3-butyric acid (IBA).

[0095] 3. Culture medium

[0096] (1) Composition and preparation method of "MS basic medium"

[0097] Table 1MS basic medium (Murashige and Skoog, 1962)

[0098] MS macroelements content MS trace element mother content Organic compound mother content <![CDATA[KNO3]]> 38 <![CDATA[MnSO4·4H2O]]> 4.46 Inositol 20 <![CDATA[NH4NO3]]> 33 <![CDATA[ZnSO4·7H2O]]> 1.72 niacin 0.1 <![CDATA[CaCl2·2H2O]]> 8.8 <![CDATA[H3BO3]]> 1.24 Vitamin B1 0.1 <![CDATA[MgSO4·7H2O]]> 7.4 KI 0.166 Vitamin B6 0.02 <![CDATA[KH2PO4]]> 3.4 <![CDATA[Na2MoO4·2H2O]]> 0.05 Glycine 0.4 <![CDATA[CuSO4·5H2O]]> 0.005 <![CDATA[CoCl2·6H2O]]> 0.005 <![CDATA[FeSO4·7H2O]]> 2.78 <![CDATA[Na2EDTA]]> 1.865

[0099] According to the required amount of culture medium, weigh agar (6 g / L) and sucrose (20 g / L), add MS minimal medium (4.43 g / L), dilute to the required volume of culture medium with sterile water, and then adjust the pH to 5.8-6.0 with 1 mol / L NaOH or 1 mol / L HCl.

[0100] (2) Differentiation medium: MS basic medium + NAA 0.005-0.05 mg / L + 6-BA 0.1-0.5 mg / L + agar 6 g / L + sucrose 30 g / L; preferably MS basic medium + NAA 0.05 mg / L + 6-BA 0.5 mg / L + agar 6 g / L + sucrose 30 g / L.

[0101] (3) Rooting medium: 1 / 2MS basic medium + NAA 0.01-0.03 mg / L + IBA 0.03-0.05 mg / L + agar 6 g / L + sucrose 30 g / L; preferably 1 / 2MS basic medium + NAA 0.02 mg / L + IBA 0.05 mg / L + agar 6 g / L + sucrose 30 g / L.

[0102] (4) Hydroponic culture medium: 1 / 2MS basic culture medium + NAA 0.01-0.03 mg / L + IBA 0.03-0.05 mg / L + sucrose 30 g / L; preferably 1 / 2MS basic culture medium + NAA 0.02 mg / L + IBA 0.05 mg / L + sucrose 30 g / L.

[0103] 4. Drug preparation

[0104] (1) 5-Aminouracil (5-AU) solution

[0105] 5-AU is dissolved in dilute hydrochloric acid, and the pH of the 5-AU solution is adjusted to 6.5-7 with sodium hydroxide, and then sterile filtered and protected from light. The concentration of the 5-AU solution is 1.1-1.7 mM, preferably 1.5 mM.

[0106] (2) Colchicine

[0107] Colchicine is prepared with distilled water to a 50 mg / L colchicine solution, which needs to be aseptically filtered and protected from light.

[0108] Except for 50 mg / L, other concentrations of colchicine, 40-60 mg / L, are suitable for the present invention.

[0109] (3) PBS

[0110] The preparation method of 1×PBS is as follows: dissolve 8.0g NaCl, 0.2g KCl, 1.44g Na2HPO4, and 0.24g KH2PO4 in distilled water, adjust the pH to 7.4, make up to 1L, sterile filter or autoclave, and store at 4°C away from light.

[0111] (4) Modified Galbraith's buffer

[0112] Modified Galbraith's buffer: 45 mM MgCl2·6H2O, 30 mM sodium citrate, 20 mM MOPS (3-morpholinepropanesulfonic acid), 0.1% (v / v) Triton X-100, and distilled water.

[0113] (5) Lysis solution

[0114] The composition of the lysis solution is: NaCl: 14.625 g, Na2EDTA: 2.92 g, Tris: 0.121 g, adjust the pH to 10, make the volume to 89 ml, and then add 1% Triton ×-100 (i.e. Triton ×-100).

[0115] Example 2 Differentiation culture and cell cycle discrimination of detached leaves of Populus argentatus 84K receptor material

[0116] 1. Differentiation culture

[0117] 1-1) Explant pretreatment

[0118] In a clean bench, take the functional leaves of the silver gland poplar 84K tissue culture seedlings grown for 4-5 weeks as explants, that is, take the leaves at the 4th or 5th leaf position from the terminal bud as explants. Use scissors to cut perpendicularly to the main vein of the leaf, forming an incision that cuts the main vein but does not cut the leaf. Usually, cut twice on each leaf, such as Figure 1 ;

[0119] 1-2) EdU incubation treatment

[0120] Then, the cut leaves were spread flat on culture dishes (9 cm in diameter) filled with solid differentiation medium for differentiation culture, wherein: EdU solution (10 μM) was added to the surface of the differentiation medium, and the thickness of the EdU solution on the surface of the differentiation medium matched the thickness of the leaves, slightly thicker than the thickness of the leaves, and slightly immersed the leaves; the culture conditions for differentiation culture were: temperature of 25±2°C; light intensity of 2000lx (usually 1500-2500lx); light cycle of 16h light / 8h dark (usually 10-16h light / 8-14h dark).

[0121] 2. Identification of cell development

[0122] Flow cytometry and EdU staining were used to instantly determine the development status of cells.

[0123] 2-1) Flow cytometry identification

[0124] 2-1A) The time starts from when the leaf is separated from the body and spread on the surface of the differentiation culture medium. During the differentiation culture treatment, the differentiated tissue at the incision of the leaf is minced with a blade in a culture dish containing 1 ml of modified Galbraith's buffer every 24 hours to prepare a leaf differentiated tissue suspension;

[0125] 2-1B) The leaf differentiated tissue suspension was filtered through a 30 μm nylon mesh, and the filtrate was stained with 100 μL DAPI (4',6-diamidino-2-phenylindole, 5 mg / mL) for 5 seconds. Ploidy analyzer (Partec PAS, Germany) was used to measure the flow cytometric histogram of the samples;

[0126] The number of cells detected in a single test was more than 10,000. The results of flow cytometry were imported into ModFit LTDNA Cell Cycle Analysis software (v.3.1.0) to analyze the changes in the proportion of cells in the G1, S, and G2 / M phases of each sample. The results are as follows: Figure 1A ; Figure 1A 1, 2, 3, and 4 represent the cell histograms measured by flow cytometry every 24 hours starting from the time when the leaves were flattened on the surface of the differentiation medium, i.e., the flow cytometric histograms at 24 hours, 48 ​​hours, 72 hours, and 96 hours of differentiation culture.

[0127] From the flow cytometric analysis, it can be seen that the proportion of S phase cells increased rapidly from 48h to 72h of differentiation culture, and G2 / M phase cells began to appear after 96h. The S phase cell growth period started 2 days after differentiation culture (48h). Figure 1A-2 ), and reached its peak after 3 days (72h) ( Figure 1A-3), and a small number of G2 / M phase cells began to appear after 4 days (96h) Figure 1A-4 ).

[0128] 2-2) EdU staining

[0129] The EdU staining method focuses on analyzing the changes in the cell cycle during the S phase.

[0130] 2-2A) Leaves cultured in differentiation medium for 24 h, 48 h, 72 h, and 96 h were subjected to EdU staining, i.e., the following treatments were performed in sequence: washing leaves with PBS for 3 times; fixing with 4% paraformaldehyde and protecting from light for 30 min; then washing with PBS for 3 times; promoting permeation with 0.5% Triton X-100; and finally performing Click-iT treatment, wherein the Click-iT treatment was strictly performed according to the EdU-Flow Cytometry Kit ( The procedure was performed according to the product instructions of 488 / 555 / 594 / 647A Click-iTEdU Flow Cytometry Assay Kits, Bioorigin, Beijing).

[0131] 2-2B) Wash the leaves twice with PBS. After removing the washing solution, cut a small amount of leaf material and place it on a slide. Add water and cover with a cover glass. Then observe under a laser confocal microscope (Leica TCS SP8; Leica, Wetzlar, Germany). The observation results are as follows: Figure 1B , cells stained with EdU emit green fluorescence, Figure 1B 1, 2, 3, and 4 represent the cell fluorescence images at 24h, 48h, 72h, and 96h of differentiation culture, respectively, starting from the time when the leaves were flattened on the surface of the differentiation medium.

[0132] The results of EdU staining were basically consistent with those of flow cytometry. The fluorescence quantity began to increase at 48 h ( Figure 1B-2 ), and at 72h the fluorescence quantity showed a dense state ( Figure 1B-3 The nuclear morphology of cells was observed under a laser confocal microscope (Leica TCS SP8; Leica, Wetzlar, Germany). Figure 1C ), during which most of the labeled nuclear morphology showed early S phase, and the cell morphology was Figure 1C-1 Differentiation culture 96h ( Figure 1B-4 ) fluorescence decreased and most of the labeled nuclei showed late S phase morphology ( Figure 1C-2 ).

[0133] Through flow cytometry and EdU staining, it was determined that the increase of S phase cells started around 48 hours of differentiation culture, the S phase peaked around 72 hours, and G2 / M phase cells began to appear around 96 hours. This laid the foundation for the subsequent design of cell cycle synchronization treatment methods. Therefore, cell cycle synchronization treatment was selected between 48-72 hours of differentiation treatment to synchronize cell differentiation and development in the S phase of the cell cycle.

[0134] Example 3 Screening of 5-AU concentration tolerance range of detached leaves of Populus argentatus 84K receptor material

[0135] Preliminary screening of 5-AU concentration tolerance range based on leaf morphological changes

[0136] 1. Differentiation culture

[0137] 1-1) Explant pretreatment

[0138] The method is the same as step 1-1) "explant pretreatment" in Example 2.

[0139] 1-2) 5-AU immersion treatment

[0140] The explant leaves were immersed in different pre-treatments with 0.5, 1, 1.5, and 2 mM of the cell cycle inhibitor 5-AU for 24 h. Figure 2 ; The leaves not soaked in 5-AU were used as the control (CK).

[0141] 1-3) Incubation

[0142] The leaves soaked in 5-AU and the leaves not soaked in 5-AU were respectively spread into a culture dish (9 cm in diameter) filled with solid differentiation culture medium for differentiation culture, wherein the culture conditions of the differentiation culture were the same as those in Example 2.

[0143] 2. Leaf morphology observation

[0144] After one week of differentiation culture, the leaf morphology was observed. Compared with the CK leaves, the leaves treated with 0.5 or 1mM 5-AU had no obvious morphological changes. Most of the leaves treated with 1.5mM 5-AU were able to differentiate normally, but a small part of them turned brown, and the whole leaf showed an uneven bumpy shape. After 2mM 5-AU treatment, the whole leaf turned brown seriously and no longer showed differentiation activity. Therefore, it was preliminarily determined that the treatment concentration of 5-AU should not exceed 2mM.

[0145] Example 3A Screening of 5-AU concentration tolerance range of detached leaves of Populus argentatus 84K receptor material

[0146] Based on the comet assay, the appropriate 5-AU concentration tolerance range of leaves was further screened, and the 5-AU concentration between 1-2mM was set to 1.1, 1.3, 1.5, 1.7, and 1.9mM for further tolerance range screening;

[0147] 1. Explant pretreatment

[0148] The method is the same as step 1-1) "explant pretreatment" in Example 2.

[0149] 2. 5-AU immersion treatment

[0150] The pretreated leaves were immersed in five different concentrations of 5-AU solutions, 1.1, 1.3, 1.5, 1.7, and 1.9 mM, for 12 h and 24 h, respectively;

[0151] 3. Comet Experiment

[0152] 3-1) The frosted glass slide is placed in a square box containing a first agarose solution (i.e., 0.6% agarose solution), and the agarose solution immerses the frosted glass slide. The 0.6% agarose solution solidifies on the surface of the frosted glass slide to form a first agarose gel layer.

[0153] 3-2) Place the leaves treated with 5-AU on ice and freeze for 10 min; cut the differentiated tissue material at the incision, add 100 μl of 1×PBS buffer, chop and shake the material with a blade, filter it through 400-mesh nylon gauze, and then absorb 30 μl of cell suspension for later use.

[0154] 3-3) Prepare 0.7% agarose solution, mix 20 μl of the cell suspension with 60 μl of the second agarose solution (i.e., 0.7% agarose solution), and spread the mixture on the first agarose layer, cover with a coverslip, and place at 4°C for 10 min to solidify.

[0155] 3-4) Place the solidified glass slide in step 3-3) in a square box, pour in 1 ml of lysis solution (NaCl: 14.625 g, Na2EDTA: 2.92 g, Tris: 0.121 g, adjust pH = 10, constant volume 89 ml, add 1% triton) pre-cooled to 4°C; and lyse in a refrigerator at 4°C in the dark for 2 h.

[0156] 3-5) After lysis, place the frosted glass slide in a new square box, add electrophoresis solution (1mmol / LEDTA, 300mmol / L NaOH) precooled to 4℃, place in a 4℃ refrigerator, and unwind for 30 minutes away from light.

[0157] 3-6) Then, electrophoresis was performed. The thawed slide was placed in an electrophoresis tank, and the electrophoresis voltage was set to 25 V for 20-30 min. After the electrophoresis, the slide was taken out of the electrophoresis tank and placed in the original square box. 0.4 mol / L Tris-HCl buffer precooled to 4°C was slowly dripped into the box, and then placed in a refrigerator for neutralization for 5 min. The neutralization step was repeated 3 times.

[0158] 3-7) Immerse the slide in anhydrous ethanol and dehydrate for 15 minutes. After the moisture on the colloid surface of the frosted slide is dried at room temperature, add ethidium bromide to the colloid surface for staining for 20 minutes.

[0159] 3-8) The DNA damage of cells after different 5-AU immersion treatments was observed under a laser confocal microscope (Leica TCS SP8; Leica, Wetzlar, Germany) under a fluorescence wavelength of 510-560 nm. The cells were observed and photographed at 40 times magnification under the microscope. The head length and the total length of the tailed cells were measured with an eyepiece micrometer, and the length of the comet tail of the cells was counted. Figure 2A , 2B , the left side is a microscope image of leaves immersed in 5-AU solution for 12 hours, and the right side is a microscope image of leaves immersed in 5-AU solution for 24 hours. When the concentration of 5-AU solution exceeds 1.7mM, the DNA tail length of cells increases significantly regardless of the treatment time ( Figure 2B ).

[0160] From the results of single cell electrophoresis, we can see that when the 5-AU concentration reaches 1.7 and 1.9 mM, the comet tail formed by DNA fragments outside the cell is more obvious ( Figure 2A-4 , 5, 9 and 10), especially when the concentration reached 1.9 mM and the treatment lasted for 24 h, the comet tail of the cells became longer and the cell body also shrank ( Figure 2A-1 0). In comparison, treatments below 1.5 mM caused less damage to cell DNA, and there was no significant difference between the treatment durations of 12 h and 24 h ( Figure 2A-1 , 2, 3, 6, 7 and 8). Therefore, the concentration of 5-AU should not exceed 1.7mM if it is suitable for subsequent experiments. 5-AU concentration ≤ 1.5mM; treatment time 12-24h. 5-AU concentration of 1.5mM or less will not cause much harm even if treated for 24h.

[0161] Example 4 Synchronous stimulation and identification of receptor materials from isolated leaves of Populus argentatus 84K

[0162] 1. Synchronous stimulation of cell cycle in vitro leaves of Populus alba 84K

[0163] 1-1) Explant pretreatment

[0164] The method is the same as step 1-1) "explant pretreatment" in Example 2.

[0165] 1-2) Pre-differentiation treatment

[0166] The pretreated leaves were spread separately into culture dishes (9 cm in diameter) filled with solid differentiation medium for pre-differentiation culture, wherein the culture conditions for pre-differentiation culture were as follows: temperature of 25±2°C; light intensity of 2000lx (usually 1500-2500lx); and light cycle of 16h light / 8h dark (usually 10-16h light / 8-14h dark).

[0167] 1-3) Capture Processing

[0168] According to the flow cytometry and EDU staining in Example 2, the development cycle of leaf bud primordium cells was determined. After 2 days (48 h) of pre-differentiation culture, that is, on the third day when the leaves were detached and flattened on the differentiation medium, different concentrations of cell cycle inhibitors (5-AU solutions) were respectively dripped into the incisions of different leaves flattened on the differentiation medium, and the capture treatment was performed under light-proof conditions, wherein: the concentrations of the 5-AU solutions were 1.1, 1.3, 1.5, and 1.7 mM, respectively, and 1±0.2 ml of the 5-AU solution was dripped into each incision of the leaves; the temperature was controlled at 25±2° C. during the capture treatment, and the capture treatment was performed for 24 h; the processing nodes and duration of the capture treatment could completely cover the growth period of the S phase cells in the induced material;

[0169] During the capture treatment, the control without adding 5-AU solution was used as CK.

[0170] 1-4) Drug release treatment

[0171] After 24 hours of light-proof capture treatment, the leaves were taken out and subjected to drug removal and release treatment, i.e., the leaves were soaked and rinsed with sterile distilled water for 4 times (usually 3-5 times).

[0172] 1-5) Recovery culture

[0173] Transfer the leaves after distilled water soaking and rinsing (i.e., drug release treatment) to a new differentiation medium, restore normal lighting, and carry out recovery culture, wherein the temperature is controlled at 25±2°C; the light intensity is 2000lx (usually 1500-2500lx); the light cycle is 16h light / 8h dark (usually 10-16h light / 8-14h dark); and the recovery culture is 1 day (usually 24-48h).

[0174] 2. Identification of the cell cycle synchronization effect of Populus alba 84K in vitro leaves

[0175] 2-1) Flow cytometry identification

[0176] The operation was the same as that of the "Flow Cytometry Identification" step in Example 2, except that the differentiated tissues at the cut of the leaves after 1 day of recovery culture treatment were minced with a blade in a culture dish containing 1 ml of modified Galbraith's buffer. The flow cytometric images of the detached leaves of Populus alba 84K treated with different concentrations of 5-AU are shown in FIG. Figure 3 , where a, b, c, and d represent flow cytometric images of 5-AU capture treatment at concentrations of 1.1, 1.3, 1.5, and 1.7 mM, respectively.

[0177] The results showed that when the concentration of 5-AU reached 1.5mM, the proportion of S phase cells in leaf bud primordium cells increased significantly. The proportion of S phase cells under this treatment (35.47%) was significantly higher than that of 1.1mM (26.19%) and 1.3mM (28.74%). However, when the concentration reached 1.7mM, a large number of cells were arrested in the S phase, and a large number of nuclear aggregates were detected ( Figure 3 -d).

[0178] Compared with the case of CK ( Figure 1A-3 , 4), the cell cycle of leaves treated with 1.5 mM 5-AU after capture and drug removal, and after 1 day of recovery culture is as follows Figure 3A -a, b, where Figure 3A -a is after drug removal treatment, Figure 3A -b is after 1 day of recovery; Figure 1A-3 Compared with 4, the number of cells in S phase and G2 / M phase increased significantly. Therefore, 5-AU at a concentration of 1.5mM can stimulate a better cell cycle synchronization effect.

[0179] Figure 3 Although the proportion of S phase cells was as high as 82.1% when 5-AU was 1.7 mM, the detection of G2 / M phase cells was 0, which means that the tissue cells were greatly affected and could not continue to differentiate well, affecting the vitality and germination rate of subsequent materials.

[0180] 2-2) EdU staining identification

[0181] The EdU staining was performed on the leaves after capture and after 1 day of recovery culture. The rest of the procedure was the same as that of “EdU staining discrimination” in Example 2. The results were as follows: Figure 3B -a, b, where a is the EdU fluorescence labeling result at the leaf incision after capture treatment; b is the EdU fluorescence labeling result at the leaf incision after 1 day of recovery culture treatment.

[0182] The observation results show that:

[0183] Compared with EdU staining of CK ( Figure 1B-3,4), the cell cycle of leaves treated with 1.5mM 5-AU solution after capture treatment, drug removal treatment, and recovery culture for 1 day is shown in Figure 3B -a, b, the results show that more and denser EdU staining was observed in the leaves treated with synchronization, and Figure 3B -b can observe a certain number of forms such as Figure 1C-3 The nuclear morphology of cells in the G2 / M phase is shown.

[0184] Combining the two methods, it was identified that the best effect of S phase synchronization stimulation was achieved by trapping with 1.5 mM 5-AU for 24 hours, and there was a significant increase in G2 / M phase cells after 24 hours of drug recovery.

[0185] The present invention applies a cell cycle inhibitor when the induction material is pre-differentiated and the cells at the incision are in the S phase growth phase of the leaf bud primordium cell cycle, so that the differentiated leaf bud primordium cells are synchronized (that is, a large number of them are in the S phase), and after drug removal, the cells enter the G2 / M phase relatively synchronously, and then a chromosome doubling inducer is applied to improve the chromosome doubling efficiency, thereby improving the plant polyploid induction efficiency.

[0186] The method of the present invention accurately and effectively determines that the cells of the plant induction material are in the growth phase of the S phase of the cell cycle and performs synchronized stimulation of the cell cycle, and the synchronized stimulation treatment covers the entire S phase of cell development; when the leaf bud primordium cells are induced to develop to the G2 / M phase, polyploidy induction is performed to achieve chromosome doubling of most leaf bud primordium cells, thereby significantly improving the efficiency, stability and repeatability of plant polyploidy induction.

[0187] Example 5 Synchronous stimulation and identification of receptor materials in the shoot apex of Populus argentatus 84K

[0188] The synchronization effect was verified by changing different materials. Since the cells in the shoot apex meristem are active frequently, the dynamic changes of the cells in the division phase can be compared, which can directly reflect the effect of synchronization on the G2 / M phase.

[0189] 1. Synchronous stimulation of cell cycle in shoot apical meristem of Populus argentatus 84K

[0190] First, the 4-5 week old tissue culture seedlings of Populus argentea 84K were transferred to a hydroponic culture medium with a 5-AU concentration of 1.5 mM and cultured in water in the dark for 24 hours, i.e., the capture treatment was performed for 24 hours, wherein the hydroponic culture medium was: 1 / 2MS basic medium + NAA 0.02 mg / L + IBA 0.05 mg / L + sucrose 30 g / L;

[0191] Next: After the drug removal treatment, the tissue culture seedlings were removed and the roots were washed three times with distilled water, i.e., the drug removal treatment was carried out;

[0192] Then: the tissue culture seedlings after drug removal treatment are transferred to a new hydroponic culture medium without 5-AU for normal light hydroponic culture, that is, recovery culture, wherein the normal light hydroponic conditions are: temperature of 25±2°C; light intensity of 2000lx (usually 1500-2500lx); light cycle of 16h light / 8h dark (usually 10-16h light / 8-14h dark).

[0193] 2. Identification of the Synchronized Stimulation Effect of the Shoot Apical Meristem of Populus argentatus 84K

[0194] 2-1) Fixation

[0195] The stem tips of normal silver gland poplar 84K tissue culture seedlings without any treatment (normal control group, CK group), the stem tips of silver gland poplar 84K tissue culture seedlings treated for 24 hours, and the stem tips of silver gland poplar 84K tissue culture seedlings sampled every 8 hours during the recovery culture were fixed, wherein during the recovery culture process, sampling was performed every 8 hours until the 56th hour of the recovery culture; the fixation method was as follows: the collected tissue culture seedling stem tip materials were pretreated with a saturated p-dichlorobenzene solution for 4 hours; then, the stem tip materials were placed in Carnoy's fixative (ethanol: acetic acid = 3:1) and fixed at 4°C for 15 hours;

[0196] 2-2) Tablet dyeing

[0197] The cell pressing staining observation method was used to observe and count the division index (MI) of the meristem cells in the shoot apex of each fixed material, wherein the specific operation steps of the cell pressing staining observation method are as follows:

[0198] 2-2A) Take out the fixed sample material and wash it with distilled water, repeat three times;

[0199] 2-2B) The washed material was placed in 1N HCl for dissociation at room temperature for 20 min;

[0200] 2-2C) The cell clusters at the growth point of the shoot apex were picked out with a dissecting needle and stained with modified phenol reagent (Carbol);

[0201] 2-2D) Place the cell cluster on a clean glass slide, cover with a coverslip, and then press to spread the cells evenly;

[0202] 2-2E) Finally, Olympus BX51 was used to observe and calculate the MI index under a 100× oil immersion objective. Three biological replicates were included at each time point, and 400-500 cells were counted in each replicate. The results are shown in Figure 2. Figure 4 , 4A .

[0203] Compared with the cells at the shoot tip of the normal control group ( Figure 4 -a), cells at the shoot tip after being captured by 5-AU for 24 h ( Figure 4 -b) Most of them were arrested in interphase, and the number of cells in mitotic phase decreased. After 40 hours of recovery from drug withdrawal, the number of cells in mitotic phase increased ( Figure 4 -c), the percentage of cells in the G2 / M phase peaked and then gradually decreased ( Figure 4A ). During the observation and statistical process, it was found that cells treated with 5-AU had the phenomenon of forming chromosome bridges ( Figure 4 -d), but this phenomenon is extremely rare. Therefore, 1.5mM 5-AU treatment for 24 hours can also stimulate the increase in the number of G2 / M phase cells in the shoot apical meristem.

[0204] Example 6 Polyploid induction based on cell cycle synchronization of detached leaves of Populus argentea 84K receptor material

[0205] 1. Cell cycle synchronization

[0206] 1-1) Explant pretreatment

[0207] The same as step 1-1) "explant pretreatment" in Example 2.

[0208] 1-2) Pre-differentiation treatment

[0209] The cut leaves were respectively spread in a culture dish (9 cm in diameter) filled with differentiation medium for pre-differentiation culture. The pre-differentiation treatment time was started from the time when the leaves were spread on the differentiation medium. The culture conditions of the pre-differentiation culture were the same as those in Example 4.

[0210] 1-3) Capture Processing

[0211] According to the flow cytometry and EDU staining in Example 2, the development cycle of leaf bud primordium cells was determined. After 2 days (48 h) of pre-differentiation culture, that is, on the third day after the leaves were spread on the differentiation medium, 5-AU solution was added to the incision of the leaves spread on the differentiation medium, and the capture treatment was performed under light-proof conditions, wherein: the concentration of the 5-AU solution was 1.5 mM (usually 1.1-1.7 mM), 1±0.2 ml of 5-AU solution was added to each incision of the leaf; the temperature was 25±2°C, and the capture treatment was performed for 24 h.

[0212] 1-4) Drug removal

[0213] After 24 hours of capture treatment, the leaves were taken out and rinsed with sterile distilled water for 3 times (usually 3-5 times), ie, drug removal treatment (ie, drug removal and release treatment) was performed.

[0214] 1-5) Recovery culture

[0215] The drug-deprived leaves were transferred to new differentiation medium, restored to normal light, and recovered culture was performed, wherein the recovery culture was the same as that in Example 4.

[0216] 1-6) Identification of cells in G2 / M phase

[0217] During the recovery culture period, EdU staining and flow cytometry were used to determine the proportion of G2 / M phase cells in the induced material after cell cycle synchronization treatment.

[0218] 1-6A) EdU staining to detect G2 / M phase cells

[0219] The procedure was the same as step 2-2) "EdU staining discrimination" of Example 2, except that EdU solution was added to the surface of the differentiation medium during the recovery culture.

[0220] The results of observations under laser confocal microscopy are as follows Figure 3B The cells stained with EdU emit green fluorescence, and most of the stained nuclei are in the G2 / M phase, shaped like Figure 1C-3 .

[0221] 1-6B) Flow cytometry to identify G2 / M phase cells

[0222] The procedure was the same as step 2-1) "flow cytometry identification" of Example 2, except that the timing was started from the time the drug-depleted leaves were transferred to the new surface of the differentiation culture medium, and during the recovery culture process, the differentiated tissue at the leaf incision was minced with a razor blade every 1 hour in a culture dish containing 1 ml of modified Galbraith's buffer to prepare a leaf differentiated tissue suspension.

[0223] From the flow cytometric analysis, we can see that: compared with the flow cytometric graph of normal differentiated leaves without cell cycle inhibitor capture treatment ( Figure 1A-3 , the peak of S phase of normal differentiated leaves), compared with the recovery culture at 0h ( Figure 3A -a) and 24h( Figure 3A -b) The proportion of cells in the S phase increased significantly, compared with the flow cytometry image of normal differentiated leaves without cell cycle inhibitor capture treatment ( Figure 1A-4 Compared with the normal differentiated leaves (G2 / M phase), the G2 / M phase cells also increased significantly.

[0224] 2. Chromosome doubling induction treatment

[0225] 2-1) Induction treatment

[0226] According to the result of step 1-6) "G2 / M phase cell identification": enter the initial phase of the G2 / M phase; add a chromosome doubling inducer (colchicine solution, 50 mg / L) to the incision of the recovery culture leaf in real time, that is, apply colchicine to the silver gland poplar 84K leaf material that has stimulated cell cycle synchronization, and perform chromosome doubling induction treatment under light-proof conditions, wherein: colchicine is a sterile colchicine solution after filtration, and the concentration is 50 mg / L; 1±0.2 ml of colchicine solution is added to each incision of the leaf; the induction treatment time is 3 days (usually 2-4 days), that is, the time starts from the addition of the colchicine solution, and the treatment is 3 days in total.

[0227] In the specific implementation manner of the present invention, the chromosome doubling inducer is taken as colchicine as an example, and other inducers are also applicable to the present invention, such as oryzalin, gibberellin, etc.

[0228] 2-2) Differentiation culture

[0229] The leaves after 3 days of induction treatment are taken out and soaked and rinsed with sterile distilled water for 3 times (usually 3-5 times); then the rinsed leaves are transferred to a new differentiation medium again, and differentiation culture is carried out under light conditions, wherein the temperature during the differentiation culture process is 25±2°C; the light intensity is 2000lx (usually 1500-2500lx); the light cycle is 16h light / 8h dark (usually 10-16h light / 8-14h dark); differentiation culture is carried out until the leaves differentiate into adventitious buds.

[0230] 2-3) Rooting culture

[0231] The obtained adventitious buds were transferred to rooting medium for rooting culture to obtain regenerated plants, and the number of regenerated plants was counted (as shown in Table 2); the temperature during the rooting culture process was 25±2°C; the light intensity was 2000lx (usually 1500-2500lx); the photoperiod was 16h light / 8h dark (usually 10-16h light / 8-14h dark).

[0232] 3. Ploidy identification of regenerated plants

[0233] 3-1) Flow cytometry identification

[0234] When the regenerated plants have grown for 3-4 weeks and their functional leaves have unfolded, ploidy identification can be performed.

[0235] The procedure was the same as step 2-1A) "flow cytometry" in Example 2, except that the leaves of the regenerated plants were cut off for flow cytometry, i.e., the leaves of the regenerated plants were chopped with a blade in a culture dish containing 1 ml of modified Galbraith's buffer to prepare a leaf tissue suspension. The results were shown in Table 2. Figure 5 ;like Figure 5-a is the diploid flow cytometry histogram, Figure 5 -b shows the tetraploid flow cytometry histogram.

[0236] 3-2) Chromosome count determination

[0237] The cell pressing staining observation method was used to observe and count the division index (MI) of the meristem cells in the shoot apex of the regenerated plants. The cell pressing staining observation method is as follows:

[0238] 3-2A) The shoot tips of the regenerated plants were pretreated with a saturated p-dichlorobenzene solution for 4 h; then, the shoot tips were placed in Carnoy's fixative (ethanol: acetic acid = 3:1) and fixed at 4°C for 15 h;

[0239] 3-2B) Take out the fixed sample material and wash it with distilled water, repeat three times;

[0240] 3-2C) The washed material was placed in 1N HCl for dissociation at room temperature for 20 min;

[0241] 3-2D) The cell clusters at the growth point of the shoot apex were picked out with a dissecting needle and stained with modified phenol reagent (Carbol);

[0242] 3-2E) Place the cell cluster on a clean glass slide, cover with a coverslip, and press firmly to spread the cells evenly.

[0243] 3-2F) Olympus BX51 was used to observe and calculate the MI index under a 100× oil immersion objective. Three biological replicates were included at each time point, and 400-500 cells were counted in each replicate.

[0244] The chromosome count results are as follows Figure 5A .like Figure 5A -a shows the diploid chromosome number, the chromosome number is 2n = 2x = 38, Figure 5A -b shows the tetraploid chromosome number, where the chromosome number is 2n=4x=76.

[0245] The chromosome count of the regenerated plants was used to obtain the number of tetraploid plants (as shown in Table 2). The induction rate was the ratio of the number of tetraploid plants detected to the total number of regenerated plants. The tetraploid induction rate is shown in Table 2.

[0246] The results of flow cytometry were consistent with those of chromosome counting.

[0247] Example 7

[0248] The process was the same as Example 6 except that the concentration of 5-AU during the capture treatment in step 1 was 1.3 mM.

[0249] Example 8

[0250] The process was the same as Example 6 except that the concentration of 5-AU during the capture treatment in step 1 was 1.7 mM.

[0251] Comparative Example 1

[0252] Except for dripping sterile distilled water onto the leaf cut during the capture process in step 1, the rest is the same as Example 6.

[0253] Comparative Example 2

[0254] Except that sterile distilled water is dripped onto the incision of the leaf blade during the capture treatment in step 1; and sterile distilled water is dripped onto the incision of the leaf blade during the induction treatment, the rest is the same as Example 6.

[0255] Table 2 Tetraploid induction based on 84K somatic cell mitosis cell cycle synchronization

[0256] Processing combination Regenerated plants Tetraploid number (strains) Tetraploid induction rate (%) Example 6 26±8.03 11.33±5.35 40.90±6.57 Example 7 33.33±2.86 3±1.41 8.60±3.56 Example 8 23±3.54 4.67±2.16 19.83±7.18 Comparative Example 1 50.67±0.82 3.67±0.82 7.26±1.68 Comparative Example 2 56 0 0

[0257] As shown in Table 2, under the synchronous stimulation of 1.5 mM 5-aminouracil, the tetraploid induction efficiency of Populus argentea 84K leaves was the most significant, with an average induction efficiency of 40.90%.

[0258] The present invention is based on the technology of synchronously stimulating the cell cycle during the differentiation process of plant induction materials under in vitro culture conditions, which effectively increases the number of M-phase cells suitable for chromosome doubling in the plant induction materials and improves the efficiency of plant polyploidy induction. The method is instant, effective and reliable.

[0259] The preferred specific embodiments of the present invention are described in detail above. It should be understood that ordinary technicians in the field can make many modifications and changes based on the concept of the present invention without creative work. Therefore, all technical solutions that can be obtained by technicians in the technical field based on the concept of the present invention through logical analysis, reasoning or limited experiments on the basis of the prior art should be within the scope of protection determined by the claims.

Claims

1. A method for improving the efficiency of plant polyploid induction based on synchronization of leaf bud primordium cell cycles, characterized in that: include: Firstly, in the differentiation culture process of sterile plant explant materials, the cell cycle inhibitor is used to perform cell development cycle synchronization stimulation treatment on leaf bud primordium cells; then the chromosome doubling inducer is used to induce treatment on the explants after the cell cycle synchronization stimulation treatment.

2. The method according to claim 1, characterized in that: The cell cycle inhibitor is 5-AU, aphidicolin or bleomycin; the chromosome doubling inducer is colchicine, oryzalin or gibberellin.

3. The method according to claim 1 or 2, characterized in that: The method also includes firstly judging the occurrence and development process of the leaf bud primordium cells of the differentiated explant material, and when the leaf bud primordium cells of the differentiated explant enter the S phase and the proportion of S phase cells begins to increase, that is, when the S phase cells are in the growth period, performing cell cycle synchronization stimulation treatment on the explant material to enrich the S phase cells.

4. A method for improving the efficiency of plant polyploid induction based on synchronization of leaf bud primordium cell cycles, characterized in that: The steps include: 1) Inoculating the explant material of the sterile plant into the differentiation medium for pre-differentiation culture; 2) To identify the developmental process of leaf bud primordium cells obtained by pre-differentiation culture of explant materials; 3) applying a cell cycle inhibitor according to the developmental process of the leaf bud primordium cells, and performing a cell development cycle synchronization stimulation treatment on the leaf bud primordium cells of the explant material, wherein the cell development cycle synchronization stimulation treatment is to use the cell cycle inhibitor to induce the leaf bud primordium cells of the explant material to stagnate in the S phase; 4) sequentially removing the drug from the explant material after the cell development cycle synchronization stimulation treatment and performing recovery culture; 5) Applying a chromosome doubling inducing agent to the explant material after recovery culture, and performing the chromosome doubling inducing treatment under light-proof conditions.

5. The method according to claim 4, characterized in that: The method for distinguishing the occurrence and development process of leaf bud primordium cells in step 2) includes: using EdU fluorescence labeling method to detect the cell proliferation dynamics of explant materials at different stages of pre-differentiation culture, and observing the changes in EdU fluorescence signals of leaf bud primordium cells entering the S phase in callus tissue through fluorescence microscopy; or using flow cytometry to detect callus tissue at different stages and analyze the distribution characteristics of the cell cycle of explant materials.

6. The method according to claim 5, characterized in that: The EdU staining test is to perform a cell development cycle synchronization excitation treatment on the explant material to enrich the S phase cells when green fluorescence is observed in the cells, indicating that the cells have developed to the S phase and the number of cells in the S phase increases (i.e., the leaf bud primordium cells in the S phase are in the growth phase); the flow cytometry test is to analyze the development cycle proportion of the leaf bud primordium cells of the callus tissue by flow cytometry, and when the proportion of the S phase cells begins to increase, i.e., when the cells are in the growth phase of the S phase, the explant material is subjected to a cell development cycle synchronization excitation treatment to enrich the S phase cells.

7. The method according to claim 4 or 5, characterized in that: The cell cycle synchronization stimulation in step 3) includes using a cell cycle inhibitor to perform cell development cycle synchronization stimulation treatment on the explant material determined to be in the S phase growth stage of the leaf bud primordium cells.

8. The method according to claim 5, characterized in that: Comet assay was used to detect the concentration and duration of cell cycle inhibitors for cell synchronization stimulation treatment.

9. The method according to claim 1 or 2, characterized in that: The chromosome doubling treatment in step 5) is to add a chromosome doubling inducer to the explant material after recovery culture, and perform the induction treatment on a differentiation medium in the dark for at least 2 days.

10. A method for obtaining polyploid regenerated plants based on synchronization of leaf bud primordium cell cycles, characterized in that: According to the method for improving the efficiency of plant polyploid induction based on synchronization of leaf bud primordium cell cycles as described in any one of claims 1 to 9, explant materials are subjected to polyploidy induction treatment; then, the explant materials after the polyploidy induction treatment are differentiated and cultured on a differentiation medium until adventitious buds are differentiated and cultured; then, the adventitious buds are inoculated on a rooting medium for rooting culture to obtain polyploid regenerated plants.

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