• 专利附录
  • 专利说明
  • 权利要求
  • 类似技术
  • 同族专利
  • 引用文献
  • 法律状态
  • 优先权
    • 专利摘要:
    Disclosed is a cryopreservation and recovery culture method for embryonic axis of Castanea mollissima. The cryopreservation of Castanea mollissima embryonic axis is carried out through dehydration treatment, loading treatment, vitrification treatment, cryopreservation treatment, thawing treatment and unloading treatment. By adopting the cryopreservation method for Castanea mollissima embryonic axis provided in the present application, the obtained Castanea mollissima embryonic axis has the feature of high germination rate.
    公开号:NL2026787A
    申请号:NL2026787
    申请日:2020-10-29
    公开日:2022-02-22
    发明作者:Wenqing Li；Xin Dong；Biao Han；Xiaoman Xie；Lu Lu；Yang Xian
    申请人:Shandong Forest Germplasm Resources Center；
    IPC主号:
    专利说明:

    [0001] [0001] The present invention relates to the technical field of plant seed (embryo) preservation, in particular to a cryopreservation and recovery culture method for embryonic axis of Castanea mollissima.BACKGROUND
    [0002] [0002] Castanea mollissima (Castanea mollissima Bl.), as a member of the genus Castanea in the family Fagaceae, is an important woody food tree species in China. The seed of Castanea mollissima is of great nutrition, and Castanea mollissima is widely distributed and planted in a large area with a high level of industrialization. Therefore, Castanea mollissima is also known as "King of Nuts" and "Iron Crops". China is the origin and main producing area of Castanea mollissima in the world, and its planting area and output are both ranked first in the world. Castanea mollissima germplasm resources are extremely rich, distributed in most provinces in the country, but because Castanea mollissima seeds are typical recalcitrant seeds, they are not resistant to dehydration and low temperature, and thus cannot be stored for a long time according to normal seed preservation methods. Cryopreservation, as an important way of long-term storage of recalcitrant seeds, will be an important direction for seed preservation of Castanea mollissima.
    [0003] [0003] At present, researches on cryopreservation of Castanea mollissima seeds in the prior art mainly focus on the effects of different freezing and thawing methods for embryonic axis of Castanea mollissima on cryopreservation. However, there has always been a problem that the germination rate of the embryonic axis after cryopreservation is low (less than 30%).
    [0004] [0004] Therefore, there is an urgent need for a cryopreservation and recovery culture method for embryonic axis of Castanea mollissima to solve the technical problem of low germination rate of embryonic axis of Castanea mollissima after cryopreservation in the prior art.SUMMARY
    [0005] [0005] The present application provides a method for cryopreservation and recovery culture for embryonic axis of Castanea mollissima, and aims to solve the technical problems of low embryonic axis germination rate of Castanea mollissima after cryopreservation and lack of a complete system of cryopreservation and recovery culture for Castanea mollissima embryonic axis in the prior art.
    [00086] [00086] In order to solve the above technical problems, an embodiment of the present application discloses the following technical solution:
    [0007] [0007] According to a first aspect of the embodiment of the present invention, a cryopreservation method for embryonic axis of Castanea mollissima comprises:
    [0008] [0008] Step 1: cutting off a bulk tissue of Castanea mollissima containing an embryonic axis, sterilizing the surface of the bulk tissue, cutting off an embryonic axis tissue for cryopreservation, and dehydrating the embryonic axis tissue;
    [0009] [0009] Step 2: putting the dehydrated embryonic axis tissue into a sterilized cryogenic tube, adding a loading liquid, and carrying out loading treatment;
    [0010] [0010] Step 3: removing the loading liquid after the loading treatment is finished, adding a pre-cooled PVS: solution, and carrying out vitrification treatment;
    [0011] [0011] Step 4: quickly putting the cryogenic tube containing the embryonic axis tissue after the vitrification treatment into a biological sample liquid nitrogen container for storage, and carrying out cryopreservation treatment;
    [0012] [0012] Step 5: carrying out thawing treatment on the embryonic axis after the cryopreservation treatment; and
    [0013] [0013] Step 6: sterilizing the cryogenic tube after the thawing treatment, exhausting the PVS: solution, adding an unloading liquid, and carrying out unloading treatment.
    [0014] [0014] Preferably, the loading liquid comprises: 2M glycerol + 0.4M sucrose, dissolved in a WPM basal medium, with the pH adjusted to 5.8.
    [0015] [0015] Preferably, the PVS: solution comprises: 30% glycerol + 15% ethylene glycol + 15% dimethyl sulfoxide + 0.4 mol/L sucrose, dissolved in a WPM basal medium, with the pH adjusted to 5.8.
    [0016] [0016] Preferably, the unloading liquid comprises: 1.2M sucrose, dissolved in a WPM basal medium, with the pH adjusted to 5.8.
    [0017] [0017] According to a second aspect of the present application, there is provided a recovery culture method for cryopreserved embryonic axis of Castanea mollissima, in which recovery culture is carried out on an embryonic axis of Castanea mollissima treated by using the cryopreservation method for embryonic axis of Castanea mollissima according to the first aspect of the present application, the method for recovery culture comprising:
    [0018] [0018] exhausting an unloading liquid after an unloading treatment of the Castanea mollissima embryonic axis is finished, adding a WPM basal medium for cleaning, exhausting the WPM basal medium, and then transferring the embryonic axis to a cryogenic recovery medium.
    [0019] [0019] Preferably, the recovery medium comprises: WPM + 1.5 mg/L 6-benzylaminopurine (6-BA) + 0.1 mg/L naphthylacetic acid (NAA) + 1 g/L polyvinylpyrrolidone (PVP) + 1 g/L activated carbon + 30 g/L sucrose + 3 g/L agar, with the pH adjusted to 5.8.
    [0020] [0020] According to a third aspect of the present application, there is provided a tissue culture medium for embryonic axis seedlings of Castanea mollissima, in which tissue culture of a Castanea mollissima seedling is carried out in the tissue culture medium, after the treatment using the cryopreservation method for Castanea mollissima embryonic axis provided in the first aspect of the present application, and after the treatment using the method for recovery culture provided in the second aspect of the present application, and the culture medium comprises: DKW + 6-BA 2 mg/L + NAA 0.2 mg/L + IBA 0.04 mg/L.
    [0021] [0021] Compared with the prior art, the present application has the beneficial effects that:
    [0022] [0022] According to the cryopreservation and recovery culture method for Castanea mollissima embryonic axis, the cryopreservation of Castanea mollissima embryonic axis is carried out through dehydration treatment, loading treatment, vitrification treatment, cryopreservation treatment, thawing treatment and unloading treatment. The procedures of loading and unloading are added before and after the procedure of cryopreservation in the prior art, so that damages caused by freezing during cryopreservation is minimized, and the germination rate of embryonic axis tissues after cryopreservation is significantly increased. In addition, a tissue culture method for embryonic axis germination is further studied, and the types of basal medium and hormone are selected and optimized, thereby obtaining an optimal tissue culture medium for Castanea mollissima embryonic axis seedlings.BRIEF DESCRIPTION OF THE DRAWINGS
    [0023] [0023] In order to more clearly illustrate the technical solution of the present application, the drawings used in the embodiments will be briefly described below, and it would be apparently for those skilled in the art to obtain other drawings according to these drawings without involving any inventive effort.
    [0024] [0024] FIG. 1(a) and FIG. 1(b) are flow charts of cryopreservation and recovery culture of Castanea mollissima embryonic axis according to an embodiment of the present invention;
    [0025] [0025] FIG. 2 is a diagram showing germination of embryonic axis with different moisture contents after cryopreservation according to an embodiment of the present invention;
    [0026] [0026] FIG. 3 is a diagram showing the effect of different concentrations of 6-BA on explant growth according to an embodiment of the present invention;
    [0027] [0027] FIG. 4 is a diagram showing the effect of different concentrations of IBA on explant growth according to an embodiment of the present invention;
    [0028] [0028] FIG. 5 is a diagram showing the effect of different concentrations of GAs on explant growth according to an embodiment of the present invention; and
    [0029] [0029] FIG. 6 is a schematic diagram of embryonic axis tissue culture according to an embodiment of the present invention.DESCRIPTION OF THE EMBODIMENTS
    [0030] [0030] For those skilled in the art to better understand the technical solutions of the present application, the technical solutions in the embodiments of the present application will be clearly and completely described in connection with the accompanying drawings in the embodiments of the present application. It is to be understood that the described embodiments are merely a part of the embodiments of the present application and are not intended to be exhaustive. Based on the embodiments of the present application, all other embodiments obtained by those skilled in the art without involving any inventive effort shall fall within the scope of the present application.
    [0031] [0031] 1. Material and method
    [0032] [0032] 1.1 Test material
    [0033] [0033] Mature seeds of Castanea mollissima (a cultivar as 'Yanquan’), cultivated in Jinan, Shandong Province, were collected in autumn, and seeds of uniform size and shape were selected and immediately sent to a germplasm preservation bank after collection for cryopreservation and recovery culture experiments. 5 [0034] 1.2 Cryopreservation and recovery culture method for Castanea mollissima embryonic axis
    [0035] [0035] FIG. 1(a) and FIG. 1{b) are flow charts of cryopreservation and recovery culture of Castanea mollissima embryonic axis according to an embodiment of the present invention. As shown in FIG. 1(a) and FIG. 1(b), a cryopreservation and recovery culture method for Castanea mollissima embryonic axis comprises the following steps:
    [0036] [0036] S11, Determination of initial moisture content.
    [0037] [0037] 20 embryonic axis tissues of uniform size were cut off and weighed to obtain a fresh weight, then the embryonic axis tissues were put into an oven (at a temperature of 103°C for 17 hours) to be dehydrated to constant weight and weighed to obtain a dry weight, wherein initial moisture content = (fresh weight - dry weight)/fresh weight x 100%, and the process is repeated for 3 times.
    [0038] [0038] S12, Preparation of embryonic axis with different moisture contents.
    [0039] [0039] To prevent damages to embryonic axis caused by sterilization, firstly, a bulk tissue (about 4 g) containing an embryonic axis were cut off, and the surface of the tissue was sterilized by soaking in 75% (v/v) alcohol for 30 s, washing with sterile water for three times, transferring to and sterilizing in a 5% sodium hypochlorite solution for 5 min, and rinsing with sterile water for three times. After the tissue was sterilized, embryonic axis tissues (about 0.2 g) for cryopreservation were cut off in a clean bench. In the sterile environment of the clean bench, the embryonic axis with different moisture contents is obtained by weight reducing method, and the embryonic axis tissues were dehydrated to moisture content gradients of 30%, 25%, 20%, 15%, 10%, etc.
    [0040] [0040] S13, Loading treatment.
    [0041] [0041] The embryonic axis tissues of Castanea mollissima with different moisture contents were put into a sterilized cryogenic tube, 2 mL of a loading liquid was added, and loading treatment was carried out for 20min. The loading liquid comprises: 2M glycerol + 0.4M sucrose, dissolved in a WPM basal medium, with the pH adjusted to 5.8.
    [0042] [0042] S14, Vitrification treatment
    [0043] [0043] After the loading treatment is finished, the loading liquid was exhausted with a pipette, 2 mL of pre-cooled on ice PVS; solution was added, and ice bath treatment was carried out for 60 min. The PVS: solution comprises: 30% glycerol + 15% ethylene glycol + 15% dimethyl sulfoxide + 0.4 mol/L sucrose, dissolved in a WPM basal medium, with the pH adjusted to 5.8.
    [0044] [0044] S15, Cryopreservation
    [0045] [0045] After the vitrification treatment is finished, the cryogenic tube was fixed on a cryogenic tube holder, and quickly put into a biological sample liquid nitrogen container for storage.
    [0046] [0046] S16, Thawing treatment
    [0047] [0047] After storage in liquid nitrogen for more than 24 hours, thawing treatment was carried out on the embryonic axis, the temperature of a water bath kettle was set to 40°C, and when the temperature was constant, the cryogenic tube was quickly inserted into the 40°C constant-temperature water bath, and thawing was carried out in the water bath for 2 min.
    [0048] [0048] S17, Unloading treatment
    [0049] [0049] The cryogenic tube after water bath was sterilized with 75% alcohol, and in the clean bench, the PVS: solution was exhausted with the pipette, 2 mL of an unloading liquid was added, and treatment was carried out for 20 min at a temperature of 25°C. The unloading liquid comprises: 1.2M sucrose, dissolved in a WPM basal medium, with the pH adjusted to 5.8.
    [0050] [0050] 1.3 Recovery culture method for cryopreserved Castanea mollissima embryonic axis
    [0051] [0051] S21, germination treatment for embryonic axis
    [0052] [0052] After the unloading treatment is finished, the unloading liquid was exhausted with the pipette, 2 mL of a WPM basal medium was added for cleaning, then the WPM basal medium was exhausted with the pipette, and then the embryonic axis tissues were transferred to a cryogenic recovery medium. The recovery medium comprises: WPM + 1.5 mg/L 6-BA + 0.1 mg/L NAA + 1 g/L PVP (polyvinylpyrrolidone) + 1 g/L activated carbon + 30 g/L sucrose + 3 g/L Agar, with the pH adjusted to 5.8.
    [0053] [0053] S31, Recovery culture
    [0054] [0054] Recovery culture was carried out in dark at a temperature of 25°C for one week, then culture was carried out in a 12 h/12 h photoperiodic environment with an ambient temperature of 25°C for one week, and at last, the germination rate was calculated. Under the same conditions, the embryonic axis tissues without cryopreservation were taken as control with 20 per group, and the process is repeated for 3 times.
    [0055] [0055] 1.4 Tissue culture test method
    [0056] [0056] Tissue culture of recovered and germinated tender stems of embryonic axis after cryopreservation was studied, and the following test method was carried out.
    [0057] [0057] (1) Orthogonal test of basal medium and hormone
    [0058] [0058] A three-level four-factor orthogonal experiment was designed with three types of basal medium such as WPM, MS, DKW and concentrations of three hormones such as 6-BA, NAA, IBA as main factors using the software Orthogonal Design Assistant V3.1. The designed orthogonal experiment is shown in Table 1. In each group, 15-20 stems were inoculated, the process was repeated for 3 times, and the growth was investigated after 28 days.
    [0059] [0059] (2) Experiment for optimization of concentration of hormone
    [0060] [0060] According to the result of the orthogonal experiment, the concentration gradient of some hormone was optimized while taking DKW as the basal medium. For all the experiments, in each group, 15-20 stems were inoculated, the process was repeated for 3 times, and the growth was investigated after 28 days..
    [0061] [0061] (1) Experiment on concentration gradient of 6-BA Table 2 Design of Test on Concentration Gradient of 6-BA __”___—_—__[_[__[_[|__„‘''„'- _|…'…__——_____ Serial number of | Basal medium 6-BA (mg/L) NAA (mg/L) IBA (mg/L) culture medium L31 DKW 1 0.2 0.02 L37 DKW 2 0.2 0.02
    [0082] [0082] (2) Experiment on concentration gradient of IBA Table 3 Design of Test on Concentration Gradient of IBA Serial number of Basal medium 6-BA (mg/L) NAA (mg/L) IBA (mg/L) culture medium L34 DKW 2 0.2 0 L37 DKW 2 0.2 0.02 L38 DKW 2 0.2 0.04
    [00863] [00863] (3) Experiment on concentration gradient of GA, Table 4 Design of Test on Concentration Gradient of GA: Le Serial number of culture Basal medium 6-BA (mg/L) GAs (mg/L) NAA (mg/L) IBA (mg/L) medium L31 DKW 1 0 0.2 0.02 L40 DKW 1 100 0.2 0.02
    [0064] [0064] (3) Investigation on tissue culture data
    [0065] [0065] After 28 days of culture, the differentiation rate, number of buds and height of buds of the seedlings were investigated, and an average number of buds and an average height were calculated. Among others: differentiation rate = number of differentiated budding stems/total number of inoculated stems, average number of buds = total number of buds /total number of inoculated stems, and average height = total height of buds/total number of buds.
    [0066] [0066] 2 Result and analysis
    [0067] [0067] 2.1 Cryopreservation method for embryonic axis
    [0068] [0068] According to the embodiments of the present invention, the cryopreservation method is optimized mainly in four aspects. (1) The loading treatment is added before cryopreservation, and correspondingly, the unloading treatment is added after cryopreservation. (2) The recovery germination medium for Castanea mollissima embryonic axis after cryopreservation is adjusted from the original improved MS medium to the improved WPM medium. (3) The preparation and pretreatment of the PVS2 solution are optimized by preparing the PVS2 solution based on a WPM liquid basal medium, while the ice bath treatment is further added. (4) The sterilization procedure of the embryonic axis tissue is carried out earlier, and damages to the embryonic axis caused by surface sterilization are minimized.
    [0069] [0069] FIG. 2 is a diagram showing germination of embryonic axis with different moisture contents after cryopreservation according to an embodiment of the present invention. With the optimization of the cryopreservation method, the result is shown in FIG. 2. As the moisture contents of the embryonic axis decreased, the germination rate of Castanea mollissima embryonic axis in the control group revealed a trend of gradual decrease. During decrease of the moisture content from 30% to 10%, the germination rate of embryonic axis in the control group decreased from 95% to 40%, indicating that the germination vigor of embryonic axis in the control group decreased gradually due to the damage of dehydration. The result of germination of the embryonic axis with different moisture contents after cryopreservation showed that the germination rate of embryonic axis decreased from 60% to 15% during the moisture content decreased from 30% to 10%, and as the moisture content decreased, the germination rate of embryonic axis after cryopreservation revealed a trend of gradual decrease, which should be resulted from damages caused by dehydration to Castanea mollissima embryonic axis. When the embryonic axis had a moisture content of 30%, the germination rate after cryopreservation was highest, i.e. 60.0%. That may be caused by three reasons: firstly, the loading liquid and the unloading liquid were added, so that the moisture state in the embryonic axis can be effectively adjusted to minimize the damage caused by freezing in the cryogenic environment; secondly, PVS: vitrification cryoprotectant prepared taking distilled water as a matrix is less effective than PVS: prepared taking a culture medium as a matrix, and the latter allows cells to be better vitrified in the cryogenic environment; and thirdly, the PVS; ice bath treatment can provide cold acclimation for the embryonic axis and induce extracellular freezing, so that the moisture content of protoplasm is reduced, and the cryogenic tolerance of plant tissues is improved.
    [0070] [0070] In summary, by using the optimized cryopreservation method, a Castanea mollissima embryonic axis can be cryopreserved without reducing the moisture content of the embryonic axis below a critical moisture content of dehydration, so that damages to the Castanea mollissima embryonic axis caused by dehydration and low temperature are prevented, and the germination rate after cryopreservation is relatively high.
    [0071] [0071] 2.2 Selection of basal medium and hormone
    [0072] [0072] According to result analysis on the range, the type of basal medium and the concentration of NAA posed the greatest effect on the differentiation rate, and significant analysis showed that the difference was significant (P <0.05), and the best effect appeared when the basal medium was WPM and the concentration of NAA was
    [0073] [0073] According to the result of the range of the orthogonal experiment, it was found that the range of the basal medium was always biggest in the three indexes as differentiation rate, average number of buds and average height, indicating that among all the factors, the basal medium posed the greatest effect on the growth indexes, and it was important to select an appropriate basal medium for tissue culture of Castanea mollissima. Three types of mediums were used in the experiment, wherein the WPM had the best effect in increasing the differentiation rate, the MS medium could result in a relatively high average number of buds, and the DKW medium resulted in the highest average height of buds. In the actual culture process, it was found that, for Castanea mollissima embryonic axis explants on the DKW medium, seedlings grew vigorously and were less vitrified with better quality, and the effect of subculture was good, so that DKW was used as the basal medium in the subsequent culture, and a hormone adjustment experiment was carried out on that basis. From the view of hormone, 6-BA had some effect on the average number of buds and average height, but not significant. The effect was greatest when 8-BA of 2 mg/L was used with, where all the three growth indexes reached the peak.
    [0074] [0074] 2. 3 Optimization of concentration of hormone
    [0075] [0075] FIG. 3 is a diagram showing the effect of different concentrations of 6-BA on explant growth according to an embodiment of the present invention. In the orthogonal experiment of basal medium and hormone, the concentration of 6-BA in the DKW medium showed different effects. When the concentration of 6-BA was 2 mg/L, the effect of promoting height growth was better than that of 6-BA of 1 mg/L, but the differentiation rate and budding rate were lower than that of 6-BA of 1 mg/L. Therefore, the two concentrations of 6-BA were re-compared to determine an optimal concentration. The result (FIG. 3) showed that when the concentration of 6-BA was 2 mg/L, the differentiation rate and the average number of buds were higher and the average height was lower. Variance analysis showed that the effect of the concentration of 6-BA on the average number of buds was significant (P <0.01), but not significant on the differentiation rate and height (P> 0.05). It indicates that the concentration of 6-BA had the greatest effect on the average number of buds, and the average number of buds directly affected the efficiency of tissue culture and rapid propagation. Therefore, 2 mg/L 6-BA was more suitable for tissue culture and rapid propagation of embryonic axis seedlings.
    [0076] [0076] FIG. 4 is a diagram showing the effect of different concentrations of IBA on explant growth according to an embodiment of the present invention. The comparison result of the concentrations of IBA (FIG. 4) showed that the effect of the concentration of IBA on the average number of buds was most significant where the number of buds revealed a trend of increase with the increase of the concentration of IBA, and the effect on the differentiation rate and the average height is smaller. When the concentration of IBA was 0.02 mg/L, the differentiation rate was highest (100%), and when the concentration of IBA was 0 mg/L, the average height was the highest (1.24 cm). Variance analysis showed that IBA had a significant effect on both the differentiation rate and the average number of buds (P < 0.01). When the concentration of IBA was 0.04 mg/L, the average number of buds was highest.
    [0077] [0077] FIG. 5 is a diagram showing the effect of different concentrations of GAs on explant growth according to an embodiment of the present invention. GAs had a certain effect on the dormancy release of Castanea mollissima seeds, and the effects of 0 mg/L and 100 mg/L GA: culture media were compared in the embodiment of the present application (FIG. 5). In the two control groups, the average number of buds and the average height of the experiment group added with GAs: were significantly lower, indicating that 100 mg/L GA; was not conducive to the growth of buds, but could improve the differentiation rate. In summary, GAs could improve the differentiation rate of tissue culture of stems, while also inhibiting buds.
    [0078] [0078] Combining the above experiment results, the optimum medium for tissue culture of Castanea mollissima embryonic axis seedlings was DKW + 6-BA 2 mg/L + NAA 0.2 mg/L + IBA 0.04 mg/L, which could ensure the quantity and quality of the seedlings. In addition, adding GAs could improve the differentiation rate.
    [0079] [0079] FIG. 6 is a schematic diagram of embryonic axis tissue culture according to an embodiment of the present invention. As shown in FIG. 6, by adopting the cryopreservation and recovery culture method for Castanea mollissima embryonic axis and the tissue culture medium for Castanea mollissima embryonic axis seedlings provided by the invention, the obtained Castanea mollissima seedlings grew vigorously.
    [0080] [0080] Since the above embodiments have been described with reference to other embodiments, and different embodiments have like parts, like or similar parts throughout the specification are made with reference to each other and are not described in detail herein.
    [0081] [0081] Other embodiments of the application will be apparent to those skilled in the art from consideration of the specification and practice of the application disclosed herein. This application is intended to cover any variations, uses, or adaptations of the application following the general principles of the application and including common general knowledge or customary technical means in the art not disclosed herein. The specification and examples should be considered as exemplary only, with a true scope and spirit of the application being indicated by the following claims.
    [0082] [0082] The above embodiments of the present application are not to be construed as limiting the scope of the present application.
    权利要求:
    Claims (7)
    [1]
    A method for cryopreservation of the embryonic axis of Castanea mollissima, comprising: Step 1: cutting a bulk tissue of Castanea mollissima containing an embryonic axis, sterilizing a surface of the bulk tissue, cutting an embryonic axis tissue for cryopreservation and dehydration of the embryonic axis tissue; Step 2: Placing the dehydrated embryonic ash tissue in a sterilized cryogenic tube, adding a loading liquid and performing a loading treatment; Step 3: removing the charging liquid after the charging treatment is completed, adding a pre-cooled PVS: solution and performing a vitrification treatment; Step 4: Rapidly placing the cryogenic tube containing the embryonic ash tissue after the vitrification treatment in a biological sample container with liquid nitrogen for storage, and performing cryopreservation treatment; Step 5: performing a thaw treatment on the embryonic axis after the cryopreservation treatment; and Step 6: sterilizing the cryogenic tube after the thawing treatment, exhausting the PVS: solution, adding a release liquid and performing a release treatment.
    [2]
    The cryopreservation method for the embryonic axis of Castanea mollissima according to claim 1, wherein the loading liquid comprises: 2 M glycerol + 0.4 M sucrose, dissolved in a WPM basal medium, with the pH adjusted to 5.8.
    [3]
    The cryopreservation method for the embryonic axis of Castanea mollissima according to claim 1, wherein the PVS: solution comprises: 30% (v/v) glycerol + 15% (v/v) ethylene glycol + 15% (v/v) dimethyl sulfoxide + 0.4 mol/L sucrose, dissolved in a WPM basal medium, with the pH adjusted to 5.8.
    [4]
    The cryopreservation method for the embryonic axis of Castanea mollissima according to claim 1, wherein the release fluid comprises: 1.2 M sucrose dissolved in a WPM basal medium, pH adjusted to 5.8.
    [5]
    A culture method for restoring cryopreserved embryonic axis of Castanea mollissima, wherein the culturing method for restoring is performed on an embryonic axis of Castanea mollissima treated by the cryopreservation method for embryonic axis of according to any one of claims 1 to 4 , and the culture recovery method comprises: exhausting a release fluid after a release treatment of the embryonic axis of Castanea mollissima is completed, adding a WPM base medium for cleaning, exhausting the WPM base medium, and then transferring the embryonic ashes to a recovery medium.
    [6]
    The culture method for recovering cryopreserved Castanea mollissima embryonic axis according to claim 5, wherein the recovery medium comprises: WPM + 1.5 mg/| 6-Benzylaminopurine (6-BA) + 0.1mg/| naphthylacetic acid (NAA) + 1 g / | polyvinylpyrrolidone (PVP) + 1g/| activated carbon + 30 g / | sucrose + 3 g/ | agar, with pH adjusted to 5.8.
    [7]
    7. A tissue culture medium for embryonic ash seedlings of Castanea mollissima, wherein the medium comprises: DKW + 6-BA 2 mg/1 + NAA 0.2 mg/| + IBA 0.04 mg/l.
    类似技术:
    公开号 | 公开日 | 专利标题
    Klimaszewska et al.2002|Conifer somatic embryogenesis: I. Development
    Bajaj1981|Regeneration of plants from potato meristems freeze-preserved for 24 months
    Baek et al.2003|Importance of explant size and origin and of preconditioning treatments for cryopreservation of garlic shoot apices by vitrification
    Leva2011|Innovative protocol for “ex vitro rooting” on olive micropropagation
    CN109757373A|2019-05-17|A kind of Jing Banxia quick breeding method for tissue culture
    Kim et al.2003|Somatic Embryogenesis from In Vitro Grown Leaf Explants of Rosa hybrida L.
    CN1775003A|2006-05-24|Method for producing detoxified sprout by cultivating garlic stem tip combined with cold treatment
    CN110352853A|2019-10-22|A method of improving Atractylis lancea test tube seedling quality and transplanting survival rate
    CN111802380B|2021-10-26|Ultralow-temperature in-vitro preservation method for stem tips of dormant buds of peaches
    Niino1995|Cryopreservation of germplasm of mulberry |
    Gupta et al.1995|Somatic embryogenesis in Douglas-fir |
    NL2026787A|2022-02-22|Cryopreservation and recovery culture for embryonic axis of castanea mollissim
    Yamada et al.1991|Cryopreservation of apical meristems of white clover |
    CN105746496B|2018-06-01|Utilize the method for vitrification ultra-low temperature Plantlet in vitro almond resting bud
    CN109984123B|2021-08-10|Ultralow temperature preservation method of pine needle brown spot resistant slash pine embryonic callus
    Tang et al.2000|Improvement of English walnut somatic embryo germination and conversion by desiccation treatments and plantlet development by lower medium salts
    Find et al.1993|Cryopreservation of an embryogenic suspension culture of Picea sitchensis and subsequent plant regeneration
    Xu et al.2009|Efficient plant regeneration in vitro from red leaf beet via organogenesis
    CN110800734A|2020-02-18|Ultra-low temperature preservation method for wild rice stem tips
    CN107223566B|2018-11-30|A kind of Wulian poplar method for tissue culture
    CN111387176B|2021-07-30|Vitrification ultralow-temperature preservation method for magnolia officinalis embryonic callus
    Reed2008|Cryopreservation of temperate berry crops
    Lu et al.2020|Establishment of an open, sugar-free tissue culture system for sugarcane micropropagation
    Vitsenia et al.2015|Effect of Explant's Size and Phytohormonal Composition of Nutritive Medium on Post-Vitrification Recovery of Garlic Meristems
    CN110583489B|2020-06-09|Tissue culture rapid propagation method and application of populus euphratica
    同族专利:
    公开号 | 公开日
    CN111685109A|2020-09-22|
    引用文献:
    公开号 | 申请日 | 公开日 | 申请人 | 专利标题
    KR20100110525A|2009-04-03|2010-10-13|대한민국|Cryopreservation methods for chrysanthemum shoot tips|
    CN110402818B|2019-08-20|2021-04-16|黄冈师范学院|Tissue culture and rapid propagation seedling raising method for mature embryos of high-quality Chinese chestnuts|
    法律状态:
    优先权:
    申请号 | 申请日 | 专利标题
    CN202010622769.2A|CN111685109A|2020-07-01|2020-07-01|Ultralow-temperature preservation and recovery culture method for Chinese chestnut hypocotyls|
    [返回顶部]