A method for preserving Panax notoginseng germplasm resources and its application

Abstract

This application relates to a method for preserving Panax notoginseng germplasm resources and its application, and relates to the field of Panax notoginseng germplasm preservation technology. The preservation method includes the following steps: (1) suspending Panax notoginseng embryogenic callus tissue in WPM medium and screening and collecting Panax notoginseng embryogenic cell clusters with a size of 0.45-1.5 mm; (2) transferring the Panax notoginseng embryogenic cell clusters into cryovials, adding loading solution for loading treatment, and obtaining loaded embryogenic cell clusters; (3) vitrifying the loaded Panax notoginseng embryogenic cell clusters in cryovials with vitrification solution to obtain vitrified Panax notoginseng embryogenic cell clusters; (4) placing the cryovials containing the vitrified Panax notoginseng embryogenic cell clusters in liquid nitrogen for preservation; after preservation, the Panax notoginseng embryogenic cell clusters are subjected to recovery culture. The survival rate after preservation by this method reaches 90%, and it can quickly differentiate into Panax notoginseng plants. The differentiated Panax notoginseng plants survive when transplanted under natural conditions, with a transplant survival rate of 90%, and grow well in the later stage.

Description

Technical Field

[0001] This application relates to the field of Panax notoginseng germplasm preservation technology, and in particular to a method for preserving Panax notoginseng germplasm resources and its application. Background Technology

[0002] Panax notoginseng (Burk.) FH Chen is a perennial herb belonging to the genus Panax in the family Araliaceae. It is mainly distributed in southwestern China and has high economic and medicinal value. As a traditional Chinese medicine, Panax notoginseng has unique and significant effects in treating cardiovascular diseases and is listed in the list of food and medicine homologous, leading to a surge in demand. However, Panax notoginseng is mainly propagated through cultivation, which has resulted in severe continuous cropping obstacles, a shrinking of its authentic producing area, and increasingly prominent pest and disease problems. Its germplasm resources face the risk of loss and degradation, requiring long-term, safe, and effective preservation methods. The seeds produced by Panax notoginseng are typically recalcitrant seeds with significant after-ripening, belonging to the morphophysiological dormancy type, and are sensitive to dehydration; therefore, conventional preservation methods are insufficient for long-term preservation.

[0003] Currently, regarding the preservation of Panax notoginseng germplasm resources, existing literature discloses one preservation method, specifically Shang Lihuang. Research on Cryopreservation of Ginseng, American Ginseng, and Panax notoginseng [D]. Jilin Agricultural University, 2018. The method describes the following: The optimal cryopreservation process for Panax notoginseng callus is as follows: Rapidly growing embryogenic callus is transferred to cryovials, and a PVS3 cryoprotectant solution (MS + 40% glycerol + 40% sucrose) is added. After treatment at room temperature for 30 minutes, the cryoprotectant is replaced with fresh cryoprotectant. The cryovial is then placed in a programmed cooling system and stored at -80℃ for 12 hours, followed by immersion in liquid nitrogen for 48 hours. Afterward, it is removed, thawed in a 40℃ water bath, and washed. After 30 days of recovery culture, the survival rate is statistically analyzed to be 80%. While this method achieves a certain degree of preservation of Panax notoginseng germplasm resources, it does not conduct further research on regenerated plants, thus failing to achieve truly effective preservation of Panax notoginseng germplasm resources. Summary of the Invention

[0004] To address or partially address the problems existing in related technologies, this application provides a method for preserving Panax notoginseng germplasm resources. By screening and collecting embryogenic cell clusters of Panax notoginseng with a size of 0.45-1.5 mm, these clusters are used as explants for cryopreservation. After conventional loading treatment, vitrification, liquid nitrogen preservation, and appropriate recovery culture, the survival rate after preservation reaches 90%. The cryopreserved Panax notoginseng embryogenic cell clusters can rapidly differentiate into Panax notoginseng plants. The differentiated Panax notoginseng plants can be transplanted and survive under natural conditions with a survival rate of 90%, exhibiting good subsequent growth. This achieves true preservation of Panax notoginseng germplasm resources.

[0005] This application discloses a method for preserving Panax notoginseng germplasm resources, comprising the following steps:

[0006] (1) In a clean bench, select Panax notoginseng embryogenic callus cultured on proliferation medium for 2 weeks under dark conditions at 25℃, and subculture for 3 weeks. The selected Panax notoginseng embryogenic callus was suspended in 150ml Erlenmeyer flasks with WPM medium, and then screened through 1.5mm and 0.45mm sieves to collect Panax notoginseng embryogenic cell clusters with a size of 0.45-1.5 mm, which were used as explants for cryopreservation.

[0007] The formulation of the proliferation medium is as follows: WPM medium, 2,4-D 2 mg / L, polyvinylpyrrolidone 1 g / L, hydrolyzed casein 1 g / L, sucrose 30 g / L, 1 g / L activated carbon and 3 g / L plant gel, with a pH of 5.8.

[0008] (2) Vitrification and cryopreservation of the Panax notoginseng embryonic cell clusters with a size of 0.45-1.5 mm obtained in step (1).

[0009] Specifically:

[0010] (a) Select Panax notoginseng embryogenic cell clusters with a size of 0.45-1.5 mm and transfer them into 2 ml cryovials. Add 2 ml of loading solution and load at room temperature for 20 minutes to obtain loaded embryogenic cell clusters. The loading solution is: WPM medium, 184 g / L glycerol and 0.4 mol / L sucrose.

[0011] (b) The loaded embryogenic cell clusters were mixed with 2 ml of PVS2 vitrification solution pretreated on ice in cryovials and vitrified on ice for 30 minutes to obtain vitrified Panax notoginseng embryogenic cell clusters; the formulation of the vitrification solution was: WPM medium, 300 g / L glycerol, 150 g / L ethylene glycol, 150 g / L dimethyl sulfoxide and 0.4 mol / L sucrose;

[0012] (c) Place cryovials containing vitrified Panax notoginseng embryonic cell clusters in liquid nitrogen for more than 24 hours;

[0013] After preservation, the embryogenic cell clusters of Panax notoginseng were recultured. Specifically:

[0014] (a) Thaw cryovials containing Panax notoginseng embryonic cell clusters in a 40°C water bath for 2 minutes after freezing them in liquid nitrogen.

[0015] (b) The thawed Panax notoginseng embryonic cell clusters were mixed with the unloading solution in the ultra-clean workbench and unloaded at room temperature for 20 minutes to obtain unloaded Panax notoginseng embryonic cell clusters.

[0016] The unloading solution was formulated as follows: WPM medium, 410.4 g / L sucrose, and pH 5.8.

[0017] (c) The unloaded Panax notoginseng embryonic cell clusters were washed twice with WPM liquid culture medium and transferred to filter paper to obtain the washed Panax notoginseng embryonic cell clusters.

[0018] (d) The cleaned Panax notoginseng embryonic cell clusters were transferred to the proliferation medium along with the filter paper for recovery culture for 4 weeks.

[0019] Subculture every two weeks, cultured in the dark at 25°C;

[0020] This embodiment also provides a method for regenerating Panax notoginseng plants based on the above-described preservation method. Specifically:

[0021] (a) Transfer the recovered embryogenic cell clusters of Panax notoginseng to a proliferation medium and culture them. Subculture them every two weeks to obtain newly established embryogenic callus of Panax notoginseng.

[0022] (b) The newly established Panax notoginseng embryogenic callus was evenly spread on the differentiation medium in the form of a thin layer to separate the Panax notoginseng somatic embryos;

[0023] The differentiation medium formula is: WPM medium, sucrose 30 g / L, plant gel 3 g / L, activated carbon 1 g / L, and the pH value of the differentiation medium is 5.8.

[0024] (c) Differentiated Panax notoginseng somatic embryos were independently selected and cultured on WPM medium with 30 g / L sucrose and 3 g / L plant gel to obtain mature Panax notoginseng somatic embryos;

[0025] (d) Transfer mature Panax notoginseng somatic embryos into tissue culture flasks for culture to obtain Panax notoginseng regenerated tissue culture seedlings;

[0026] (e) After transplanting and acclimatizing the regenerated tissue culture seedlings of Panax notoginseng in a greenhouse, regenerated Panax notoginseng plants are obtained.

[0027] Compared with the prior art, the beneficial effects of this application are:

[0028] 1. The vitrification cryopreservation technique can be used to promote the long-term, safe, and effective preservation of Panax notoginseng embryogenic callus, achieving a survival rate of up to 90%. Compared to existing preservation methods, the embryogenic cell clusters regenerated through this cryopreservation method exhibit strong recovery capabilities. Figure 3 ), short recovery period ( Figure 3 ( ), which can achieve rapid and large-scale proliferation in a short period of time.

[0029] 2. After cryopreservation, the embryogenic cell clusters of Panax notoginseng can rapidly differentiate into Panax notoginseng plants. These differentiated plants can be transplanted and survive under natural conditions, with a survival rate of up to 90%, and exhibit good growth in the later stages. This achieves true preservation of Panax notoginseng germplasm resources. Attached Figure Description

[0030] The above and other objects, features and advantages of this application will become more apparent from the more detailed description of exemplary embodiments thereof in conjunction with the accompanying drawings, wherein the same reference numerals generally represent the same components in the exemplary embodiments thereof.

[0031] Figure 1 These are schematic diagrams illustrating the morphology of Panax notoginseng embryonic cell clusters with different pore sizes.

[0032] in, Figure 1 In the diagram, a, b, c, d, and e are morphological images of embryonic cell masses with different pore sizes under a stereomicroscope, while f, g, h, i, and j are morphological images of embryonic cell masses with different pore sizes in 20 ml of WPM medium. In a and f, the pore size is less than 100 μm; in b and g, the pore size is 100-200 μm; in c and h, the pore size is 200-450 μm; in d and i, the pore size is 450-1000 μm; and in e and j, the pore size is greater than 1000 μm.

[0033] Figure 2 This is a schematic diagram showing the effect of different vitrification solutions and different treatment times on the regeneration rate of Panax notoginseng embryonic cell clusters during cryopreservation. Figure 2 a, b, c, d, e, and f in the table are used to indicate the significance of differences between groups;

[0034] Figure 3 This is a schematic diagram comparing the proliferation rates of Panax notoginseng embryonic cells re-established after cryopreservation and those that have undergone cryopreservation.

[0035] Figure 4 This is a schematic diagram of the process by which embryonic cells of Panax notoginseng recover and develop into seedlings;

[0036] in, Figure 4 In the middle, a is a schematic diagram of Panax notoginseng embryogenic callus after two weeks of proliferation culture;

[0037] b is a schematic diagram of selected Panax notoginseng embryonic cell clusters of 0.45-1.5 mm;

[0038] c is a schematic diagram of cell regeneration after one week of recovery culture following cryopreservation of Panax notoginseng embryonic cell clusters;

[0039] d is a schematic diagram of embryonic cells re-established through proliferation culture of regenerated cell clusters;

[0040] e is a schematic diagram of the cotyledonary embryo undergoing regeneration of embryogenic cells;

[0041] f is a schematic diagram of germinating cotyledonary embryos;

[0042] g is a schematic diagram of a Panax notoginseng somatic cell seedling with germinating cotyledonary embryos;

[0043] h is a schematic diagram of transplanted, survived, cryopreserved, and regenerated 2-year-old embryos. Detailed Implementation

[0044] The embodiments of this application will now be described in more detail with reference to the examples. While embodiments of this application are shown in the examples, it should be understood that this application can be implemented in various forms and should not be limited to the embodiments set forth herein. Rather, these embodiments are provided to make this application more thorough and complete, and to fully convey the scope of this application to those skilled in the art.

[0045] Example 1

[0046] The method for preserving Panax notoginseng germplasm resources in this embodiment consists of the following steps:

[0047] 1. The zygotic embryos of mature Panax notoginseng seeds were induced and cultured in an induction medium (cultured in the dark at 25℃) for 4 consecutive months, with the medium being changed every month to obtain Panax notoginseng embryogenic callus tissue.

[0048] The formulation of the induction medium is as follows: WPM medium, 2,4-D 2 mg / L, 6-BA 0.5 mg / L, polyvinylpyrrolidone 1 g / L, hydrolyzed casein 1 g / L, sucrose 30 g / L and plant gel 3 g / L, with a pH of 5.8.

[0049] 2. In a clean bench, add WPM medium to a sterilized centrifuge tube. Use tweezers to pick up the well-grown Panax notoginseng embryogenic callus obtained in step 1, transfer it to a centrifuge tube, suspend the Panax notoginseng embryogenic callus in WPM medium, and then pass it through 1.5 mm and 0.45 mm sieves to collect Panax notoginseng embryogenic cell clusters with a size of 0.45-1.5 mm.

[0050] Figure 1 Schematic diagram of the morphology of Panax notoginseng embryonic cell clusters with different pore sizes;

[0051] 3. Loading treatment: Transfer the Panax notoginseng embryonic cell clusters into cryovials, add loading solution for loading treatment, and obtain loaded embryonic cell clusters; the loading solution is formulated as follows: WPM medium, 184 g / L glycerol and 0.4 mol / L sucrose;

[0052] 4. Vitrification: The loaded Panax notoginseng embryonic cell clusters are vitrified in cryovials with a vitrification solution to obtain vitrified Panax notoginseng embryonic cell clusters.

[0053] The vitrification solution was formulated as follows: WPM medium, 300 g / L glycerol, 150 g / L ethylene glycol, 150 g / L dimethyl sulfoxide, and 0.4 mol / L sucrose.

[0054] 5. Ultra-low temperature preservation: After vitrification, fix the cryovial containing the vitrified Panax notoginseng embryonic cell clusters onto the cryovial holder, and then quickly place the cryovial holder into a liquid nitrogen tank for preservation for more than 24 hours.

[0055] 6. Thawing in a 40°C water bath: After being stored in liquid nitrogen for more than 24 hours, remove the cryopreservation rack with the attached cryovial from the liquid nitrogen tank and quickly insert it into a 40°C constant temperature water bath for thawing for 2 minutes.

[0056] 7. Unloading process: After the cryovials have been thawed in a water bath, wipe the outer surface with 75% alcohol, remove the vitrification solution with a pipette in a clean bench, add 2 mL of unloading solution, and treat at 25°C for 20 min.

[0057] The unloading solution was formulated with WPM medium and 410.4 g / L sucrose, and the pH of the unloading solution was 5.8.

[0058] 8. Cleaning and Recovery Culture: After unloading, aspirate the unloading solution with a pipette, add 2 mL of WPM medium for cleaning, and then aspirate the WPM liquid again with a pipette. After cleaning, add 1 mL of WPM medium to a 2 mL cryovial, attach a pipette tip with a slit, and transfer the Panax notoginseng embryogenic cell mass to a 9 cm glass culture dish containing 3 sheets of filter paper. After the filtrate has completely permeated the cell mass, transfer it to the induction medium described in step 1 and perform recovery culture at 25°C in the dark.

[0059] 9. Identification of survival rate and recovery ability, and selection of optimal vitrification solution treatment time.

[0060] After thawing and culturing the Panax notoginseng embryonic cell clusters for two weeks, the cells that show a pale yellow color and show proliferation are considered alive, while those that are all white or black and show no proliferation are considered dead.

[0061] The regeneration rate was calculated based on the survival rate of the Panax notoginseng embryonic cell clusters in each cryopreservation tube. FDA staining and direct observation yielded consistent statistical results.

[0062] The most accurate time point for determining the survival rate of Panax notoginseng embryonic cell clusters is 48 hours of recovery culture.

[0063] Thirty tubes of Panax notoginseng embryogenic cell clusters frozen for two weeks were recultured. Five cryovials were used in each experiment, and six experiments were conducted. Two consecutive experiments were counted as one replicate, for a total of three replicates. Ten tubes were recultured in each of the first, second, and third replicates. The results showed an average survival rate of 90% after reculture. Furthermore, cell cluster regeneration was observed in each cryovial, and the regeneration rate was 100% for 15 cryovials.

[0064] Based on observations of Panax notoginseng embryogenic cell clusters preserved at ultra-low temperatures after vitrification for 10, 20, 30, and 40 minutes after two weeks of recovery culture (thawed in a water bath on recovery medium), it was determined that 30 minutes was the optimal vitrification time for Panax notoginseng embryogenic cell clusters, and the optimal vitrification solution was PVS2 (…). Figure 2 ).

[0065] Figure 2 This is a schematic diagram showing the effect of different vitrification solutions and different treatment times on the regeneration rate of Panax notoginseng embryonic cell clusters during cryopreservation.

[0066] 10. Subculture, differentiation, and seedling formation of Panax notoginseng embryonic cell mass

[0067] After one month of recovery culture, the regenerated Panax notoginseng embryogenic cell clusters on filter paper were transferred to an induction medium without filter paper (the induction medium was similar to that in step 1, the only difference being the sucrose concentration of 30 g / L), and subcultured at 25°C in the dark. The medium was changed every two weeks. After two subcultures, the Panax notoginseng embryogenic cell clusters subcultured for two weeks were selected and transferred to WPM medium to allow differentiation. After one month, somatic embryos were produced without callus tissue. The somatic embryos were picked out and placed on WPM medium. Once roots and cotyledons began to grow, they were transferred to tissue culture flasks for further culture. After the root system was well developed, they were transplanted into seedling holes and finally made into bag seedlings. The survival rate of the bag seedlings after transplanting exceeded 90%.

[0068] Its recovery and seedling formation process is as follows Figure 4 As shown, Figure 4 Image a shows a schematic diagram of Panax notoginseng embryogenic callus after two weeks of proliferation culture; image b shows a schematic diagram of selected Panax notoginseng embryogenic cell clusters of 0.45-1.5 mm; image c shows a schematic diagram of cell cluster regeneration after one week of cryopreservation and recovery culture; image d shows a schematic diagram of embryogenic cells re-established through proliferation culture of regenerated cell clusters; image e shows a schematic diagram of cotyledonary embryos differentiated from regenerated embryogenic cells; image f shows a schematic diagram of germinating cotyledonary embryos; image g shows a schematic diagram of Panax notoginseng somatic cell seedlings with germinating cotyledonary embryos; image h shows a schematic diagram of transplanted, survived, cryopreserved, and regenerated two-year-old somatic cell seedlings.

[0069] Comparative Example 1

[0070] Compared with Example 1, the difference in Comparative Example 1 is that different diameter cell clusters of Panax notoginseng embryogenic callus were screened when the callus was suspended in WPM medium; all other aspects were the same (subsequent loading, vitrification, liquid nitrogen preservation, and recovery culture were consistent). The regeneration capacity of cell clusters of corresponding diameters on proliferation medium was measured.

[0071] Experimental results show that the regeneration capacity is only 0.5% for 100μm, 2.6% for 100-200μm, 38.75% for 200-450μm, and 100% for 450-1500μm.

[0072] Explants with a size of 1500μm or larger are not suitable for cryopreservation because they contain a large number of undifferentiated somatic embryos (somatic embryos are difficult to regenerate into plants after cryopreservation).

[0073] Instead of sieving through 1.5mm and 0.45mm sieves to obtain Panax notoginseng embryogenic cell clusters, Panax notoginseng embryogenic callus was directly selected from the suspension. All other procedures were the same (subsequent loading, vitrification, preservation in liquid nitrogen, and recovery culture were all consistent).

[0074] As can be seen from the results of Comparative Example 1 and Example 1, it is essential to select Panax notoginseng embryogenic cell clusters with a size of 0.45-1.5 mm for cryopreservation of Panax notoginseng embryogenic callus.

[0075] The proliferation rate of Panax notoginseng embryonic cells in Comparative Example 1 was measured and compared with the proliferation rate of Panax notoginseng embryonic cells re-established without cryopreservation. The results are as follows: Figure 3 As shown;

[0076] from Figure 3 As can be seen from the data, the proliferation rate of Panax notoginseng embryonic cells re-established after cryopreservation in this application was significantly higher than that of the control group that did not undergo cryopreservation.

[0077] The various embodiments of this application have been described above. These descriptions are exemplary and not exhaustive, nor are they limited to the disclosed embodiments. Many modifications and variations will be apparent to those skilled in the art without departing from the scope and spirit of the described embodiments. The terminology used herein is chosen to best explain the principles, practical application, or improvement of the technology in the market, or to enable others skilled in the art to understand the embodiments disclosed herein.

Claims

1. A method for preserving Panax notoginseng germplasm resources, characterized in that, Includes the following steps: (1) The embryogenic callus of Panax notoginseng was suspended in WPM medium and then screened through sieves with a size of 1.5 mm and 0.45 mm to collect Panax notoginseng embryogenic cell clusters with a size of 0.45-1.5 mm, which were used as explants for cryopreservation. The embryogenic callus of Panax notoginseng was prepared by the following method: the zygotic embryo of mature Panax notoginseng seeds was continuously induced and cultured in an induction medium at 25°C in the dark for 4 months, and the medium was changed once a month to obtain the embryogenic callus of Panax notoginseng. The formulation of the induction medium is as follows: WPM medium, 2,4-D 2 mg / L, 6-BA 0.5 mg / L, polyvinylpyrrolidone 1 g / L, hydrolyzed casein 1 g / L, sucrose 30 g / L and plant gel 3 g / L, with a pH of 5.

8. (2) Transfer the Panax notoginseng embryonic cell clusters into cryovials, add loading solution for loading treatment, and obtain loaded embryonic cell clusters; during loading treatment, the loading solution volume is 2 ml, the number of Panax notoginseng embryonic cell clusters is 10-40, the loading treatment temperature is 20-25 ℃, and the loading treatment time is 20-40 min. The loading solution was formulated as follows: WPM medium, 184 g / L glycerol and 0.4 mol / L sucrose; (3) The loaded Panax notoginseng embryonic cell clusters were vitrified in cryovials with vitrification solution to obtain vitrified Panax notoginseng embryonic cell clusters; the vitrification temperature was 0℃ and the vitrification time was 5-40min. The vitrification solution was formulated as follows: WPM medium, 300 g / L glycerol, 150 g / L ethylene glycol, 150 g / L dimethyl sulfoxide, and 0.4 mol / L sucrose. (4) Place the cryovial containing the vitrified Panax notoginseng embryonic cell mass in liquid nitrogen for preservation; after preservation, perform recovery culture on the Panax notoginseng embryonic cell mass.

2. The method for preserving Panax notoginseng germplasm resources according to claim 1, characterized in that, The liquid nitrogen is stored for at least 24 hours.

3. The method for preserving Panax notoginseng germplasm resources according to claim 1, characterized in that, The specific operation of the recovery culture in step (4) is as follows: (a) Thaw cryovials containing Panax notoginseng embryonic cell clusters in a 40°C water bath for 2 minutes after freezing them in liquid nitrogen. (b) The thawed Panax notoginseng embryonic cell clusters were mixed with the unloading solution in the ultra-clean workbench and unloaded at room temperature for 20 minutes to obtain unloaded Panax notoginseng embryonic cell clusters. The unloading solution was formulated as follows: WPM medium, 410.4 g / L sucrose, and pH 5.

8. (c) The unloaded Panax notoginseng embryonic cell clusters were washed twice with WPM medium and transferred to filter paper to obtain the washed Panax notoginseng embryonic cell clusters. (d) Transfer the cleaned Panax notoginseng embryonic cell clusters to proliferation medium for recovery culture.

4. The application of the method for preserving Panax notoginseng germplasm resources according to claim 1 or 3 in the regeneration of Panax notoginseng plants, characterized in that, Includes the following steps: (a) Transfer the recovered embryogenic cell clusters of Panax notoginseng to a proliferation medium and culture them. Subculture them every two weeks to obtain newly established embryogenic callus of Panax notoginseng. (b) The newly established Panax notoginseng embryogenic callus was evenly spread on the differentiation medium in the form of a thin layer to separate the Panax notoginseng somatic embryos; The differentiation medium formula is: WPM medium, sucrose 30 g / L, plant gel 3 g / L, activated carbon 1 g / L, and the pH value of the differentiation medium is 5.

8. (c) Differentiated Panax notoginseng somatic embryos were independently selected and cultured on a medium formulated with WPM medium, 30 g / L sucrose and 3 g / L plant gel to obtain mature Panax notoginseng somatic embryos. (d) Transfer mature Panax notoginseng somatic embryos into tissue culture flasks for culture to obtain Panax notoginseng regenerated tissue culture seedlings; (e) After transplanting and acclimatizing the regenerated tissue culture seedlings of Panax notoginseng in a greenhouse, regenerated Panax notoginseng plants are obtained.

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