Preservation method and application of panax notoginseng germplasm resources

Abstract

The invention relates to a preservation method and application of panax notoginseng germplasm resources, and relates to the technical field of panax notoginseng germplasm preservation. The preservation method comprises the following steps: (1) suspending pseudo-ginseng embryogenic calluses and a WPM culture medium, and screening and collecting to obtain pseudo-ginseng embryogenic cell clusters with the size of 0.45-1.5 mm; (2) transferring the panax notoginseng embryogenic cell cluster into a cryopreservation tube, and adding a loading solution for loading treatment to obtain a loaded embryogenic cell cluster; (3) carrying out vitrification treatment on the loaded panax notoginseng embryogenic cell cluster and a vitrification solution in a cryopreservation tube to obtain a vitrified panax notoginseng embryogenic cell cluster; (4) placing the cryopreservation tube filled with the vitrified pseudo-ginseng embryogenic cell cluster in liquid nitrogen for preservation; and after preservation, carrying out recovery culture on the pseudo-ginseng embryogenic cell cluster. According to the method, the survival rate reaches 90% after preservation, pseudo-ginseng plants can be rapidly differentiated, the differentiated pseudo-ginseng plants survive after being transplanted under natural conditions, the transplanting survival rate reaches 90%, and the pseudo-ginseng plants grow well in the later period.

Description

Technical Field

[0001] The present application relates to the technical field of Panax notoginseng germplasm preservation, and in particular to a method for preserving Panax notoginseng germplasm resources and applications thereof. Background Art

[0002] Panax notoginseng (Burk.) FH Chen is a perennial herb of the genus Panax in the Araliaceae family. It is mainly distributed in southwest China and has high economic and medicinal value. As a traditional Chinese medicine, Panax notoginseng has unique effects and significant therapeutic effects in the treatment of cardiovascular diseases. It has been included in the catalog of medicine and food homology, which has led to a sharp increase in its demand. However, Panax notoginseng is mainly propagated in the form of cultivated species, with serious obstacles to continuous cropping, a narrowing of the authentic production area, and increasingly prominent problems with pests and diseases. Its germplasm resources are facing the risk of loss and degradation, and its germplasm resources need a long-term, safe and effective preservation method. The seeds produced by Panax notoginseng are typical stubborn seeds with obvious after-ripening properties. They belong to the morphological and physiological dormancy type and are sensitive to dehydration. Therefore, conventional preservation methods are difficult to preserve for a long time.

[0003] At present, regarding the preservation of Panax notoginseng germplasm resources, a preservation method has been disclosed in the existing literature. The specific document is Shang Lihuang. Research on ultra-low temperature preservation of ginseng, American ginseng and Panax notoginseng [D]. Jilin Agricultural University, 2018. It also discloses the following content: The optimal ultra-low temperature preservation process of Panax notoginseng callus is: after the embryonic callus of Panax notoginseng in the rapid growth period is transferred into a cryopreservation tube, a cryoprotectant PVS3 solution (MS+40% glycerol+40% sucrose) is added, and the cryoprotectant is replaced after being treated at room temperature for 30 minutes. The cryoprotectant is placed in a program cooling instrument, placed in a -80℃ refrigerator for 12 hours, and then put into liquid nitrogen for 48 hours. Take it out, thaw it in a 40℃ water bath, and wash it. After 30 days of recovery culture, the statistical survival rate is 80%. Although this method has achieved the preservation of Panax notoginseng germplasm resources to a certain extent, it has not conducted further research on regenerated plants, and therefore cannot achieve truly effective preservation of Panax notoginseng germplasm resources. Summary of the invention

[0004] In order to solve or partially solve the problems existing in the related art, the present application provides a method for preserving Panax notoginseng germplasm resources, by screening and collecting Panax notoginseng embryonic cell clusters with a size of 0.45-1.5 mm, as cryopreserved explants, and then after conventional loading treatment, vitrification treatment, preservation in liquid nitrogen and corresponding recovery culture, it is shown that the survival rate after preservation is 90%, and the Panax notoginseng embryonic cell clusters after cryopreservation can be quickly differentiated into Panax notoginseng plants, and the differentiated Panax notoginseng plants can be transplanted and survived under natural conditions, and the transplantation survival rate is 90%, and the later growth is good. Thereby realizing the preservation of Panax notoginseng germplasm resources in a true sense.

[0005] A method for preserving Panax notoginseng germplasm resources in the present application comprises the following steps: (1) In a clean bench, select embryonic callus of Panax notoginseng that has been cultured on proliferation medium at 25℃ in the dark for 2 weeks, with a subculture period of 3 weeks. Suspend the selected embryonic callus of Panax notoginseng with WPM medium in a 150ml Erlenmeyer flask, and then pass through 1.5mm and 0.45mm sieves to collect embryonic cell clusters of Panax notoginseng with a size of 0.45-1.5 mm as explants for ultra-low temperature preservation.

[0006] The formula of the proliferation culture medium is: WPM culture medium, 2 mg / L 2,4-D, 1 g / L polyvinyl pyrrolidone, 1 g / L hydrolyzed casein, 30 g / L sucrose, 1 g / L activated carbon and 3 g / L plant gel, and the pH value of the proliferation culture medium is 5.8; (2) The Panax notoginseng embryonic cell clusters with a size of 0.45-1.5 mm obtained by screening in step (1) are vitrified and ultra-low-temperature preserved.

[0007] Specifically: (a) selecting embryonic cell clusters of Panax notoginseng with a size of 0.45-1.5 mm and transferring them into a 2 ml cryopreservation tube, adding 2 ml of loading solution and performing loading treatment at room temperature for 20 minutes to obtain loaded embryonic cell clusters; the loading solution comprises: WPM culture medium, 184 g / L glycerol and 0.4 mol / L sucrose; (b) mixing the loaded embryonic cell mass with 2 ml of PVS2 vitrification solution pre-treated on ice in a cryopreservation tube and performing vitrification treatment on ice for 30 minutes to obtain vitrified Panax notoginseng embryonic cell mass; the formula of the vitrification solution is: WPM culture medium, 300 g / L glycerol, 150 g / L ethylene glycol, 150 g / L dimethyl sulfoxide and 0.4 mol / L sucrose; (c) placing the cryovial containing the vitrified Panax notoginseng embryonic cell mass in liquid nitrogen for more than 24 hours; After preservation, the embryonic cell mass of Panax notoginseng is restored and cultured. Specifically: (a) Thaw the cryotube containing embryonic cell clusters of Panax notoginseng frozen in liquid nitrogen in a 40°C water bath for 2 minutes; (b) mixing the thawed Panax notoginseng embryonic cell mass with the unloading solution in an ultra-clean workbench, and performing an unloading treatment at room temperature for 20 minutes to obtain an unloaded Panax notoginseng embryonic cell mass; The formula of the unloading solution is: WPM medium, sucrose 410.4 g / L, and the pH value of the unloading solution is 5.8; (c) After unloading, the 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.

[0008] (d) The washed Panax notoginseng embryonic cell clusters together with the filter paper were transferred to a proliferation culture medium for recovery culture for 4 weeks.

[0009] Subculture was performed every two weeks, and the culture conditions were 25°C in the dark; This embodiment also provides a method for regenerating Panax notoginseng plants based on the above preservation method. Specifically: (a) transferring the recovered embryonic cell mass of Panax notoginseng to a proliferation medium for culture, and subculturing once every two weeks to obtain reestablished embryonic callus of Panax notoginseng; (b) applying the re-established embryonic callus of Panax notoginseng evenly in the form of a thin layer on a differentiation medium to obtain differentiated Panax notoginseng somatic embryos; The formula of differentiation medium is: WPM medium, sucrose 30g / L, phytogel 3g / L, activated carbon 1g / L, and the pH value of differentiation medium is 5.8; (c) The differentiated Panax notoginseng somatic embryos were independently selected and cultured on WPM medium + sucrose 30 g / L + phytogel 3 g / L to obtain mature Panax notoginseng somatic embryos; (d) transferring the mature Panax notoginseng somatic embryos to a tissue culture bottle for culture to obtain Panax notoginseng regeneration tissue culture seedlings; (e) After the Panax notoginseng regenerated tissue culture seedlings are transplanted and domesticated in a greenhouse, Panax notoginseng regenerated plants are obtained.

[0010] Compared with the prior art, the beneficial effects of this application are: 1. The embryonic callus of Panax notoginseng can be preserved safely and effectively for a long time through the ultra-low temperature preservation technology of vitrification, and the survival rate after preservation is 90%. Compared with the preservation methods in the prior art, the embryonic cell clusters regenerated after ultra-low temperature preservation in this method have strong recovery ability ( Figure 3 ), short recovery period ( Figure 3 ), which can achieve short-term large-scale proliferation.

[0011] 2. The embryonic cell clusters of Panax notoginseng preserved at ultra-low temperatures can quickly differentiate into Panax notoginseng plants. The differentiated Panax notoginseng plants can be transplanted and survive under natural conditions, with a transplant survival rate of 90% and good growth in the later stage. This realizes the true preservation of Panax notoginseng germplasm resources. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] The above and other objects, features and advantages of the present application will become more apparent through a more detailed description of exemplary embodiments of the present application in conjunction with the accompanying drawings, wherein the same reference numerals generally represent the same components in the exemplary embodiments of the present application.

[0013] Figure 1 This is a schematic diagram of the morphology of Panax notoginseng embryonic cell clusters with different pore sizes; in, Figure 1 a, b, c, d, e are morphological pictures of embryonic cell clusters with different pore sizes under a stereomicroscope, and f, g, h, i, j are morphological pictures of embryonic cell clusters with different pore sizes in 20 ml WPM medium; the pore size in a and f is less than 100 μm; the pore size in b and g is 100-200 μm; the pore size in c and h is 200-450 μm; the pore size in d and i is 450-1000 μm; the pore size in e and j is greater than 1000 μm; Figure 2 This is a schematic diagram showing the effects of different vitrification solution treatments 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 are used to indicate the significance of differences between groups; Figure 3 This is a schematic diagram comparing the proliferation rates of Panax notoginseng embryonic cells that have not been cryopreserved and have been reestablished after cryopreservation; Figure 4 This is a schematic diagram of the process of Panax notoginseng embryonic cells recovering into seedlings; in, Figure 4 Middle a is a schematic diagram of embryonic callus of Panax notoginseng after two weeks of proliferation culture; b is a schematic diagram of the selected 0.45-1.5 mm embryonic cell clusters of Panax notoginseng; c is a schematic diagram of the regeneration of Panax notoginseng embryonic cell clusters after one-week recovery culture after cryopreservation; d is a schematic diagram of embryonic cells reestablished by proliferation culture of regenerated cell clusters; e is a schematic diagram of the cotyledon embryo that differentiates from the regenerated embryonic cells; f is a schematic diagram of the germinating cotyledon embryo; g is a schematic diagram of Panax notoginseng somatic embryo seedlings with germinating cotyledon embryo development; h is a schematic diagram of transplanted survival and regeneration of somatic embryo seedlings preserved for 2 years. DETAILED DESCRIPTION

[0014] The embodiments of the present application will be described in more detail below with reference to examples. Although the embodiments of the present application are shown in the examples, it should be understood that the present application can be implemented in various forms and should not be limited by the embodiments set forth herein. On the contrary, these embodiments are provided to make the present application more thorough and complete, and the scope of the present application can be fully communicated to those skilled in the art.

[0015] Example 1 A method for preserving Panax notoginseng germplasm resources in this embodiment comprises the following steps: 1. Induce and culture the zygotic embryos of mature seeds of Panax notoginseng in an induction medium (cultured in the dark at 25°C) for 4 consecutive months, changing the medium every 1 month to obtain embryonic callus of Panax notoginseng; The formula of the induction medium is: WPM medium, 2 mg / L 2,4-D, 0.5 mg / L 6-BA, 1 g / L polyvinyl pyrrolidone, 1 g / L hydrolyzed casein, 30 g / L sucrose and 3 g / L plant gel, and the pH value of the induction medium is 5.8.

[0016] 2. In a clean bench, add WPM culture medium to a sterilized centrifuge tube. Use tweezers to pick up the embryonic callus of Panax notoginseng obtained in step 1 with good growth status, transfer it to a centrifuge tube, suspend the embryonic callus of Panax notoginseng with WPM culture medium, and then pass it through 1.5 mm and 0.45 mm sieves respectively to screen and collect the embryonic cell clusters of Panax notoginseng with a size of 0.45-1.5 mm; Figure 1 Schematic diagram of the morphology of Panax notoginseng embryonic cell clusters with different pore sizes; 3. Loading treatment: the embryonic cell mass of Panax notoginseng is transferred into a cryopreservation tube, and a loading solution is added for loading treatment to obtain a loaded embryonic cell mass; the formula of the loading solution is: WPM culture medium, 184 g / L glycerol and 0.4 mol / L sucrose; 4. Vitrification treatment: vitrify the loaded Panax notoginseng embryonic cell mass with a vitrification solution in a cryopreservation tube to obtain a vitrified Panax notoginseng embryonic cell mass; The formula of the vitrification solution is: WPM culture medium, 300 g / L glycerol, 150 g / L ethylene glycol, 150 g / L dimethyl sulfoxide and 0.4 mol / L sucrose; 5. Ultra-low temperature storage: After the vitrification treatment is completed, fix the cryotube containing the vitrified Panax notoginseng embryonic cell cluster on the cryotube holder, and then quickly put the cryotube holder into the liquid nitrogen tank for storage for more than 24 hours.

[0017] 6. Thawing in a 40℃ water bath: After being stored in liquid nitrogen for more than 24 hours, take out the cryotube holder from the liquid nitrogen tank, quickly insert it into a 40℃ constant temperature water bath, and thaw in the water bath for 2 minutes.

[0018] 7. Unloading treatment: After the cryopreserved tubes are thawed in a water bath, wipe the outer surface with 75% alcohol, use a pipette to suck out the vitrification solution in the clean bench, then add 2 mL of unloading solution and treat at 25°C for 20 minutes; The formula of the unloading solution is: WPM culture medium and sucrose 410.4 g / L, and the pH value of the unloading solution is 5.8.

[0019] 8. Washing and recovery culture: After the unloading treatment is completed, use a pipette to suck out the unloading solution, add 2mL WPM culture medium to wash, and then use a pipette to suck out the WPM liquid. After washing, add 1mL WPM culture medium to the 2mL cryopreservation tube, use a pipette equipped with a cut tip, transfer the embryonic cell mass of Panax notoginseng to a 9cm glass culture dish with 3 filter papers, and transfer the filtrate to the induction culture medium described in step 1 for recovery culture at 25°C in the dark.

[0020] 9. Identification of survival rate and recovery ability and selection of optimal vitrification solution treatment time After the thawed Panax notoginseng embryonic cell clusters are restored to culture for two weeks, if the proliferation of light yellow Panax notoginseng embryonic cells is observed, it means that the cells are alive, while if all the cells are white or black and there is no proliferation, they are dead.

[0021] The regeneration rate was calculated based on the survival of the embryonic cell clusters of Panax notoginseng in each cryopreserved tube. The statistical results of FDA staining and direct observation were consistent.

[0022] The most accurate time point for determining the survival rate of Panax notoginseng embryonic cell clusters was 48 hours after recovery culture, thus concluding that 48 hours was the most accurate time point for determining the survival rate of Panax notoginseng embryonic cell clusters.

[0023] 30 tubes of embryonic cell clusters of Panax notoginseng that had been frozen for two weeks were recultured, with 5 cryopreserved tubes per experiment, 6 experiments, and 2 consecutive experiments were counted as 1 replicate, for a total of 3 replicates, of which 10 tubes were recultured each time for the 1st, 2nd, and 3rd replicates. The results showed that the average survival rate after reculture was 90%. Further, according to the calculation of cryopreserved tubes, cell clusters were regenerated in each cryopreserved tube, and the regeneration rate of 15 cryopreserved tubes was 100%.

[0024] Combined with the observation of the ultra-low temperature storage of Panax notoginseng embryonic cell clusters after 10, 20, 30, and 40 minutes of vitrification treatment during two weeks of recovery culture (thawing in a water bath on recovery medium), it was concluded that 30 minutes was the optimal vitrification treatment time for Panax notoginseng embryonic cell clusters, and the optimal vitrification solution was PVS2 ( Figure 2 ).

[0025] Figure 2 This is a schematic diagram showing the effects of different vitrification solution treatments and different treatment times on the regeneration rate of Panax notoginseng embryonic cell clusters during cryopreservation; 10. Subculture, differentiation and seedling formation of embryonic cell clusters of Panax notoginseng After one month of resuming culture, transfer the embryonic cell clusters of Panax notoginseng regenerated on the filter paper to the induction medium without filter paper (the induction medium is similar to the induction medium in step 1, the only difference is that the concentration of sucrose is 30g / L), and perform subculture at 25°C in the dark. Change the culture medium every two weeks. After two subcultures, select the embryonic cell clusters of Panax notoginseng that have been subcultured for two weeks and transfer them to WPM medium for differentiation. After one month, somatic embryos are produced without callus tissue. The somatic embryos are picked out and placed on WPM medium. When the roots and cotyledons begin to grow, they are transferred to tissue culture bottles for further culture. After the root system is well developed, they are moved to the seedling hole and finally made into bag seedlings; the survival rate of bag seedlings after transplanting exceeds 90%.

[0026] The recovery process is as follows Figure 4 As shown, Figure 4 a is a schematic diagram of Panax notoginseng embryonic callus tissue that has been proliferated and cultured for two weeks; b is a schematic diagram of the selected 0.45-1.5mm Panax notoginseng embryonic cell clusters; c is a schematic diagram of the regeneration of Panax notoginseng embryonic cell clusters after ultra-low temperature preservation and recovery culture for one week; d is a schematic diagram of the embryonic cells re-established by proliferation culture of the regenerated cell clusters; e is a schematic diagram of the cotyledon embryos differentiated from the regenerated embryonic cells; f is a schematic diagram of the germinated cotyledon embryos; g is a schematic diagram of Panax notoginseng somatic embryo seedlings with germinated cotyledon embryos; h is a schematic diagram of the transplanted somatic embryo seedlings that survived ultra-low temperature preservation and regenerated for 2 years.

[0027] Comparative Example 1 Compared with Example 1, the difference of Comparative Example 1 is that when the embryonic callus of Panax notoginseng is suspended with the WPM medium, the embryonic cell clusters of Panax notoginseng with different diameters are selected, and the rest are the same (the subsequent loading treatment, vitrification treatment, storage in liquid nitrogen and recovery culture are all the same). The regeneration ability of the cell clusters with corresponding diameters on the proliferation medium is determined.

[0028] The experimental results show that the regeneration capacity of 100μm is only 0.5%, the regeneration rate of 100-200μm is 2.6%, the regeneration rate of 200-450μm is 38.75%, and the regeneration rate of 450-1500μm is 100%.

[0029] Explants larger than 1500 μm in size are not suitable for cryopreservation because they contain a large number of undifferentiated somatic embryos (it is difficult to form regenerated plants after somatic embryos are cryopreserved).

[0030] The embryonic cell mass of Panax notoginseng was not obtained by sieving through 1.5 mm and 0.45 mm sieves, but the embryonic callus of Panax notoginseng was directly selected from the suspension, and the rest were the same (the subsequent loading treatment, vitrification treatment, storage in liquid nitrogen and recovery culture were all the same).

[0031] It can be seen from the results of Comparative Example 1 and Example 1 that when cryopreserving the embryonic callus of Panax notoginseng, it is necessary to select the embryonic cell clusters of Panax notoginseng with a size of 0.45-1.5 mm for cryopreservation.

[0032] The proliferation rate of the embryonic cells of Panax notoginseng in Comparative Example 1 was measured and compared with the proliferation rate of the embryonic cells of Panax notoginseng reestablished without cryopreservation. The results are as follows: Figure 3 As shown; from Figure 3 It can be seen that the proliferation rate of Panax notoginseng embryonic cells re-established after ultra-low temperature preservation in the present application is significantly higher than that of the control group that has not been ultra-low temperature preserved.

[0033] The embodiments of the present application have been described above, and the above description is exemplary, not exhaustive, and is not limited to the disclosed embodiments. Many modifications and changes will be apparent to those of ordinary skill in the art without departing from the scope and spirit of the described embodiments. The selection of terms used herein is intended to best explain the principles of the embodiments, practical applications, or improvements to the technology in the market, or to enable other persons of ordinary skill in the art to understand the embodiments disclosed herein.

Claims

1. A method for preserving Panax notoginseng germplasm resources, characterized in that: The following steps are involved: (1) The embryonic callus of Panax notoginseng was suspended with WPM medium, and then passed through 1.5 mm and 0.45 mm sieves respectively to screen and collect the embryonic cell clusters of Panax notoginseng with a size of 0.45-1.5 mm as explants for ultra-low temperature preservation; (2) transferring the Panax notoginseng embryonic cell mass into a cryopreservation tube, adding a loading solution for loading treatment, and obtaining a loaded embryonic cell mass; The formula of the loading solution is: WPM culture medium, 184 g / L glycerol and 0.4 mol / L sucrose; (3) vitrifying the loaded Panax notoginseng embryonic cell mass with a vitrification solution in a cryopreservation tube to obtain a vitrified Panax notoginseng embryonic cell mass; The formula of the vitrification solution is: WPM culture medium, 300 g / L glycerol, 150 g / L ethylene glycol, 150 g / L dimethyl sulfoxide and 0.4 mol / L sucrose; (4) The cryopreservation tube containing the vitrified Panax notoginseng embryonic cell mass is placed in liquid nitrogen for storage; after storage, the Panax notoginseng embryonic cell mass is restored for culture.

2. A method for preserving Panax notoginseng germplasm resources according to claim 1, characterized in that: The embryonic callus of Panax notoginseng in step (1) is prepared by the following preparation method: zygotic embryos of mature seeds of Panax notoginseng are continuously induced and cultured in an induction medium at 25° C. in the dark for 4 months, and the medium is changed every 1 month, so as to obtain the embryonic callus of Panax notoginseng; The formula of the induction medium is: WPM medium, 2 mg / L 2,4-D, 0.5 mg / L 6-BA, 1 g / L polyvinyl pyrrolidone, 1 g / L hydrolyzed casein, 30 g / L sucrose and 3 g / L plant gel, and the pH value of the induction medium is 5.

8.

3. A method for preserving Panax notoginseng germplasm resources according to claim 1, characterized in that: During the loading treatment in step (2), the volume of the loading solution is 2 ml, the number of clusters of Panax notoginseng embryonic cells is 10-40, the loading treatment temperature is 20-25° C., and the loading treatment time is 20-40 min.

4. A method for preserving Panax notoginseng germplasm resources according to claim 1, characterized in that: The temperature of the vitrification treatment in step (3) is 0°C, and the time of the vitrification treatment is 5-40 minutes.

5. A method for preserving Panax notoginseng germplasm resources according to claim 1, characterized in that: The storage time in liquid nitrogen is at least 24 hours.

6. A 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 the cryotube containing embryonic cell clusters of Panax notoginseng frozen in liquid nitrogen in a 40°C water bath for 2 minutes; (b) mixing the thawed Panax notoginseng embryonic cell mass with the unloading solution in an ultra-clean workbench, and performing an unloading treatment at room temperature for 20 minutes to obtain an unloaded Panax notoginseng embryonic cell mass; The formula of the unloading solution is: WPM medium, sucrose 410.4 g / L, and the pH value of the unloading solution is 5.8; (c) The unloaded Panax notoginseng embryonic cell mass was washed twice with WPM medium and transferred onto filter paper to obtain the washed Panax notoginseng embryonic cell mass; (d) The washed Panax notoginseng embryonic cell clusters were transferred to proliferation medium for recovery culture.

7. Use of a method for preserving Panax notoginseng germplasm resources according to claim 1 or 6 in regeneration of Panax notoginseng plants, characterized in that: The following steps are involved: (a) transferring the recovered embryonic cell mass of Panax notoginseng to a proliferation medium for culture, and subculturing once every two weeks to obtain reestablished embryonic callus of Panax notoginseng; (b) applying the re-established embryonic callus of Panax notoginseng evenly in the form of a thin layer on a differentiation medium to obtain differentiated Panax notoginseng somatic embryos; The formula of differentiation medium is: WPM medium, sucrose 30g / L, phytogel 3g / L, activated carbon 1g / L, and the pH value of differentiation medium is 5.8; (c) The differentiated Panax notoginseng somatic embryos were independently selected and cultured on a medium containing WPM medium, 30 g / L sucrose and 3 g / L phytagel to obtain mature Panax notoginseng somatic embryos; (d) transferring the mature Panax notoginseng somatic embryos to a tissue culture bottle for culture to obtain Panax notoginseng regeneration tissue culture seedlings; (e) After the Panax notoginseng regenerated tissue culture seedlings are transplanted and domesticated in a greenhouse, Panax notoginseng regenerated plants are obtained.

Images