Method for cultivating clustered seedlings based on direct induction of apricot cotyledon embryo removal
By using the decotyledon embryo of apricot as an explant, the clump buds were directly induced and the clump buds were expanded and expanded, the problems such as difficulty in induction of clump buds in apricot tissue culture were solved, and efficient reproduction effect was achieved, with a proliferation coefficient reaching 20.4.
Patent Information
- Application Number
- CN202510035973.7
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-01-09
- Publication Date
- 2025-04-15
AI Technical Summary
There are problems in apricot tissue culture with difficulty in induction of cluster buds, poor quality of bud bushes, low proliferation coefficient and long expansion and reproduction cycle.
The decotyledon embryo is used as the explant to directly induce the generation of cluster buds, and the cluster seedlings are formed through elongation and culture, and the cluster bud induction medium of specific components and proportions and management parameters such as light and temperature are used to achieve efficient cluster bud induction.
The reproduction efficiency of tissue culture is greatly improved, and the proliferation coefficient can reach 20.4, simplifying the operation steps, reducing the probability of errors, and shortening the culture time.
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Figure CN119817466A_ABST
Abstract
Description
Technical Field
[0001] The invention belongs to the technical field of tissue culture, and in particular relates to a method for directly inducing and cultivating clustered seedlings based on apricot cotyledon-free embryos. Background Art
[0002] Apricot is mainly distributed in northern my country. It is a major economic tree species and ecological greening tree species with wide application and practical value. At present, the main propagation methods of apricot are seed propagation and grafting propagation. Among them, grafting propagation can maintain the excellent characteristics of the variety and stabilize the genetic traits, but the propagation speed is slow; seed propagation requires stratification and other technologies, the seed processing time is long and the requirements are high, and the seed yield is often affected by the climate.
[0003] As a new technology, tissue culture technology uses plant organs or tissues as explants, dedifferentiates to form callus tissue, and then redifferentiates to form adventitious buds, and finally develops into a complete plant. It can reproduce plants with stable genetic traits in a short period of time, and is not affected by region, climate, etc. Cluster buds are group cluster buds developed from plant organs or callus tissue. Most of them are multi-cellular, with complete structure, can directly form plants, and have a high seedling rate.
[0004] However, in the process of apricot tissue culture, there are problems such as difficulty in inducing cluster buds, poor bud cluster quality, low proliferation coefficient, and long propagation cycle. Summary of the invention
[0005] In order to solve the problems existing in the above-mentioned prior art, the present invention provides a method for directly inducing and cultivating cluster seedlings based on cotyledonless apricot embryos. The method uses the cotyledonless embryos as explants to directly induce the production of cluster buds, forms cluster seedlings through elongation culture, and propagates through division. The proliferation coefficient can reach 20.4, which greatly improves the efficiency of tissue culture reproduction.
[0006] The specific technical solution adopted by the present invention is:
[0007] A method for directly inducing and cultivating clustered seedlings based on apricot cotyledon-free embryos, comprising the following steps:
[0008] S1. removing the seed shell of an apricot kernel and retaining the kernel, then peeling off the seed coat of the kernel and removing the cotyledon to obtain a cotyledon-free embryo with a complete plumule, radicle, and hypocotyl structure;
[0009] S2, inoculating the decotyledon embryos into a cluster bud induction medium and culturing until clusters of buds are produced on the embryonic axes of the decotyledon embryos;
[0010] S3, transferring the bud clusters in the bud induction medium to a growth medium for ramet culture to obtain a plurality of bud seedlings;
[0011] S4. After culturing the seedlings for 12-15 days, transfer them to rooting medium to induce rooting.
[0012] Furthermore, the seed kernels described in step S1 are disinfected and then soaked in sterile water, and then peeled and cotyledons are removed under sterile conditions.
[0013] Furthermore, the disinfection treatment comprises the following steps: soaking the seed kernels in ethanol for 3-6 minutes, taking them out and rinsing them with sterile water, then soaking them in disinfectant water for 10-20 minutes, taking them out and rinsing them again with sterile water;
[0014] The disinfectant water contains 0.1%-0.15% by volume of Tween-20 and 1.5%-2.0% by volume of sodium hypochlorite.
[0015] Furthermore, the clustered shoot induction medium described in step S2 uses MS medium as the basic medium and also includes 1.0-2.0 mg / L 6-BA and 0.75 mg / L NAA.
[0016] Furthermore, the culture conditions of the decotyledon embryos in the cluster bud induction medium in step S2 include: the decotyledon embryos are illuminated for 14-18 hours per day, the light intensity is 1500-2500 lux, and the culture temperature is 22-28°C.
[0017] Furthermore, the method for inoculating the decotyledon embryo into the cluster bud induction medium is: inserting the radicle of the decotyledon embryo downward into the cluster bud induction medium, and exposing the embryo upward outside the cluster bud induction medium.
[0018] Furthermore, the ramet culture described in step S3 specifically includes the following steps:
[0019] S301, transferring the bud clusters in the bud cluster induction medium to the growth medium A for cultivation, and when the individual bud cluster seedlings on the bud clusters are 2.8-3.2 cm tall and have grown 5-7 leaves, cutting the individual bud seedlings from the base of the bud clusters;
[0020] S302, transferring the cut single sprouts to growth medium B for cultivation;
[0021] S303. The seedlings that do not meet the standards on the bud cluster are subcultured to growth medium C for further cultivation, and are divided after reaching the growth standards, until all the seedlings in the bud cluster are divided.
[0022] Furthermore, the growth medium A, growth medium B, and growth medium C have the same components. The growth medium A, growth medium B, and growth medium C all use MS medium as the basic medium and also include 0.4-0.6 mg / L 6-BA and 0.3-0.5 mg / L IBA.
[0023] Furthermore, the rooting medium described in step S4 uses 1 / 2MS medium as the basic medium and also includes 0.4 mg / L IBA and 0.2 mg / L NAA.
[0024] Furthermore, the variety of apricot kernels selected is No. 17 for kernel use.
[0025] The beneficial effects of the present invention are:
[0026] 1. The present invention uses cotyledon-free embryos as explants to directly induce the production of clustered buds, which are then elongated to form clustered seedlings. The proliferation coefficient can reach 20.4 by dividing and propagating, which greatly improves the efficiency of tissue culture propagation.
[0027] In the prior art, stem segments are generally used to propagate axillary buds to produce clustered buds, but the explant contamination rate is high and the clustered bud proliferation coefficient is low; or explants such as mature embryos are used to induce callus tissue, and clustered buds are induced through redifferentiation, which involves the steps of callus induction, redifferentiation to form adventitious buds, adventitious bud proliferation, etc., and the proliferation coefficient is low. In the present invention, the cotyledons of the peeled seed kernels are removed, a wound is formed at the hypocotyl, and the cell layer with division and growth characteristics at the wound is used to directly induce the formation of clustered buds, and the proliferation coefficient is high.
[0028] 2. The steps in the present invention are simple, which reduces the operation steps and thus reduces the probability of errors in the cultivation process.
[0029] Most of the current tissue culture techniques require the use of explants to induce callus tissue, and then the callus tissue is subcultured and differentiated to induce adventitious buds, and then expanded. However, in the present invention, the cotyledons of the peeled seed kernels are removed, a wound is formed at the hypocotyl, and the cell layer with division and growth characteristics at the wound is used to directly induce the generation of clustered buds, which reduces the steps, shortens the culture time, and makes the process simpler. In order to achieve the direct induction of almond cotyledon-free embryos, the present invention adopts a clustered bud induction medium (MS+1.0-2.0mg / L 6-BA+0.75mg / L NAA) with specific components and ratios, as well as management parameters such as light and temperature. The combination of the two can achieve the direct induction of almond cotyledon-free embryos. BRIEF DESCRIPTION OF THE DRAWINGS
[0030] Figure 1 For explants: cotyledon-free embryos;
[0031] Figure 2 The inoculation direction of the cotyledon-free embryos on the cluster shoot induction medium;
[0032] Figure 3 On the 22nd day, a large number of bud clusters were produced on the hypocotyl;
[0033] Figure 4The clump seedlings were inoculated before the ramet culture on the 48th day;
[0034] Figure 5 For the division culture of cluster seedlings;
[0035] Figure 6 This is the 12th day of adventitious root induction. DETAILED DESCRIPTION
[0036] The present invention will be further described below in conjunction with the accompanying drawings and specific embodiments:
[0037] The Tween-20 described in the present invention is polyoxyethylene sorbitan monolaurate, 6-BA is 6-benzylaminopurine, NAA is naphthylacetic acid, and IBA is indolebutyric acid.
[0038] Example 1
[0039] S1. Select dried apricot kernel No. 17 harvested in the current year, remove the seed shell and keep the seed kernel for disinfection, soak the disinfected seed kernel in sterile water for 12 hours, then peel off the seed coat under sterile conditions, remove the cotyledon to obtain the cotyledon-free embryo with complete plumule, radicle and hypocotyl structure, such as Figure 1 As shown;
[0040] The disinfection method is as follows: in a clean bench, soak the seed kernels in 75% ethanol for 5 minutes, take them out and rinse them with sterile water for 3 times, then soak them in 2.0% sodium hypochlorite solution with 0.1% Tween-20 added for 15 minutes, take them out and rinse them with sterile water for 3 times;
[0041] S2. Inoculate the decotyledon embryos into a cluster bud induction medium for cultivation. The inoculation method is to insert the decotyledon embryos with the radicle facing downward into the medium, with the plumule facing upward and exposed outside the medium. Figure 2 As shown, the culture conditions were 16 hours / day, light intensity of 2000 lux, and temperature of 25°C, and the decotyledonated embryos were cultured until bud clusters formed on the embryonic axis;
[0042] The cluster bud induction medium uses MS medium as the basic medium and also includes 1.0 mg / L 6-BA and 0.75 mg / L NAA;
[0043] S3, culture to 28 days, transfer the bud cluster in the culture medium to growth medium A for culture, continue to culture for 20 days, when the individual clump seedlings on the bud cluster are 3 cm high and have grown 6 leaves, start ramet culture, cut the individual bud seedlings from the base of the bud cluster, and then transfer them to growth medium B for culture, subculture the clump seedlings that do not meet the standards to growth medium C for further culture until all the clump seedlings of the bud cluster are rameted;
[0044] Among them, the growth medium A, B, and C have the same composition, all of which use MS medium as the basic medium and also include 0.5 mg / L 6-BA and 0.4 mg / L IBA;
[0045] S4. After 14 days of culturing the seedlings, they are transferred to the rooting medium to induce rooting. After 10-14 days, 2-4 taproots can be observed growing in the seedlings.
[0046] The rooting medium uses 1 / 2MS medium as the basic medium and also includes 0.4 mg / L IBA and 0.2 mg / L NAA.
[0047] Embodiment 2-4
[0048] The difference between Example 2-4 and Example 1 is only the different ratios of the clustered shoot induction culture medium. The specific ratios are shown in Table 1.
[0049] Embodiment 5-8
[0050] The difference between Examples 5-8 and Example 1 is only the different components or proportions of the rooting medium, as shown in Table 2 for details.
[0051] Comparative Examples 1-4
[0052] The difference between Comparative Examples 1-4 and Example 1 is only that the components of the clustered bud induction medium are different. In the clustered bud induction medium in Comparative Examples 1-4, TDZ (thidiazuron) is used to replace 6-BA. The specific components are shown in Table 1.
[0053] Comparative Examples 5-7
[0054] The difference between Comparative Examples 5-7 and Example 1 is that the varieties of apricot kernels are different. Comparative Examples 5-7 respectively select Weixuan No. 1, Renyong No. 3 and Jizaohong, as shown in Table 3 for details.
[0055] 2. Performance Testing
[0056] 1. The methods of Examples 1-4 and Comparative Examples 1-4 were used to induce cluster buds of decotyledonous embryos. Kernel apricot No. 17 seed core was selected. 30 explants were treated in each group. The decotyledonous embryos were cultured in cluster bud induction medium until the 22nd day, and then the cluster bud induction rate of each group was statistically analyzed. The test results are shown in Table 1.
[0057] Table 1
[0058]
[0059]
[0060] As can be seen from Table 1, the direct induction of the cotyledonless embryos in Examples 1-3 not only resulted in the embryos developing into seedlings, but also produced a large number of bud clusters at the hypocotyls, while in Example 4 and Comparative Examples 1-4, although the embryos developed into seedlings and there were a small amount of callus at the radicle, there were no bud clusters.
[0061] Among them, the induction rate of Example 3 was the highest, and MS+2.0mg / L 6-BA+0.75mg / L NAA was confirmed to be the best medium. The bud clusters generated in Example 3 were transferred to the elongation growth medium and cultured for 20 days. Figure 4 As shown, the ramet culture was then performed, as Figure 5 As shown, after all the division work was completed, the average proliferation coefficient of a bud cluster was calculated to be 20.4, that is, a bud cluster can expand into an average of 20.4 apricot seedlings.
[0062] 2. The seedlings obtained by the ramet culture in Example 3 were induced to root using the rooting medium in Example 3 (0.4 mg / L IBA and 0.2 mg / L NAA) and Examples 5-8, respectively. Thirty seedlings were treated in each group, and the rooting conditions were observed after 12 days. See Table 2 for details.
[0063] Table 2
[0064] deal with IBA(mg / L) NAA(mg / L) Rooting rate (%) Average number of roots Example 3 0.4 0.2 95.0 3.7 Example 5 0.3 - 55.6 2.0 Example 6 0.6 - 46.7 2.0 Example 7 0.9 - 66.7 2.8 Example 8 0.5 0.3 77.8 2.3
[0065] As can be seen from Table 2, the rooting medium used in Example 3 has 0.4 mg / L IBA and 0.2 mg / L NAA, and its rooting rate is as high as 95.0%, and the average number of roots is 3.7. Figure 6 shown.
[0066] In Examples 5-7, NAA was not added, and the rooting rate was significantly lower. Although the rooting rate increased with the increase in the concentration of IBA, it was still less than 70%, and the number of roots was also low. In Example 8, although IBA and NAA were used in combination, the rooting rate was improved, but due to the high concentrations of the two, the average number of roots decreased.
[0067] It can be seen from this that only by using the specific concentrations of IBA and NAA in combination in this application can a better rooting effect be achieved on apricot seedlings.
[0068] 3. The comparative examples 5-7 were respectively subjected to cluster bud induction of cotyledonless embryos by the method of Example 3. The composition of the cluster bud induction medium was MS+2.0mg / L 6-BA+0.75mg / L NAA. The comparative examples 5-7 respectively adopted Weixuan No. 1, Renyong No. 3, and Jizaohong seed core. 30 explants were processed in each group. The cotyledonless embryos were cultured in the cluster bud induction medium for 22 days, and then the cluster bud induction rate of each group was statistically analyzed. The test results are specifically shown in Table 3.
[0069] Table 3
[0070]
[0071] As can be seen from Table 3, the cotyledon-free embryos of different apricot varieties can all be induced to produce clustered buds using the method of Example 3, but there are large differences in the induction rates, among which the induction rate of No. 17 for kernel use is the highest. Therefore, the methods and parameters in this application are mainly aimed at No. 17 for kernel use.
Claims
1. A method for directly inducing and cultivating clustered seedlings based on apricot cotyledon embryos, characterized in that: The following steps are involved: S1, removing the seed shell of the apricot kernel and retaining the kernel, then peeling off the seed coat of the kernel and removing the cotyledon to obtain a cotyledon-free embryo with a complete plumule, radicle, and hypocotyl structure; S2, inoculating the decotyledon embryos into a cluster bud induction medium and culturing until the embryonic axes of the decotyledon embryos produce clusters of buds; S3, transferring the bud clusters in the bud induction medium to a growth medium for ramet culture to obtain a plurality of bud seedlings; S4. After culturing the seedlings for 12-15 days, transfer them to rooting medium to induce rooting.
2. A method for directly inducing and cultivating clustered seedlings based on apricot cotyledon embryos according to claim 1, characterized in that: The seed kernels described in step S1 are disinfected and then soaked in sterile water, and then peeled and the cotyledons are removed under sterile conditions.
3. A method for directly inducing and cultivating clustered seedlings based on apricot cotyledon embryos according to claim 2, characterized in that: The disinfection treatment comprises the following steps: soaking the seed kernels in ethanol for 3-6 minutes, taking them out and rinsing them with sterile water, then soaking them in disinfectant water for 10-20 minutes, taking them out and rinsing them again with sterile water; The disinfectant water contains 0.1%-0.15% by volume of Tween-20 and 1.5%-2.0% by volume of sodium hypochlorite.
4. A method for directly inducing and cultivating clustered seedlings based on apricot cotyledon embryos according to claim 1, characterized in that: The clustered shoot induction medium described in step S2 uses MS medium as the basic medium and also includes 1.0-2.0 mg / L 6-BA and 0.75 mg / L NAA.
5. A method for directly inducing and cultivating clustered seedlings based on apricot cotyledon-free embryos according to claim 1, characterized in that: The culture conditions of the decotyledon embryos in the cluster bud induction medium in step S2 include: the decotyledon embryos are illuminated for 14-18 hours per day, the light intensity is 1500-2500 lux, and the culture temperature is 22-28°C.
6. The method for directly inducing and cultivating cluster seedlings based on apricot cotyledon-free embryos according to claim 1, characterized in that: The inoculation method of the decotyledon embryo into the cluster bud induction medium is as follows: inserting the radicle of the decotyledon embryo downward into the cluster bud induction medium, and exposing the embryo upward outside the cluster bud induction medium.
7. The method for directly inducing and cultivating cluster seedlings based on apricot cotyledon-free embryos according to claim 1, characterized in that: The ramet culture described in step S3 specifically comprises the following steps: S301, transferring the bud clusters in the bud cluster induction medium to the growth medium A for cultivation, and when the individual bud cluster seedlings on the bud clusters are 2.8-3.2 cm tall and have grown 5-7 leaves, cutting the individual bud seedlings from the base of the bud clusters; S302, transferring the cut single sprouts to growth medium B for cultivation; S303. The seedlings that do not meet the standards on the bud cluster are subcultured to growth medium C for further cultivation, and are divided after reaching the growth standards, until all the seedlings in the bud cluster are divided.
8. A method for directly inducing and cultivating clustered seedlings based on apricot cotyledon-free embryos according to claim 7, characterized in that: The growth medium A, growth medium B and growth medium C have the same components. The growth medium A, growth medium B and growth medium C all use MS medium as the basic medium and also include 0.4-0.6 mg / L 6-BA and 0.3-0.5 mg / L IBA.
9. The method for directly inducing and cultivating cluster seedlings based on apricot cotyledon-free embryos according to claim 1, characterized in that: The rooting medium described in step S4 uses 1 / 2MS medium as the basic medium and also includes 0.4 mg / L IBA and 0.2 mg / L NAA.
10. The method for directly inducing and cultivating cluster seedlings based on apricot cotyledon-free embryos according to claim 1, characterized in that: The variety of apricot kernel selected is No. 17 for kernel use.