Method for in vitro conservation of agapanthus praecox l. germplasm and application thereof

Abstract

The application relates to the technical field of germplasm resource protection and utilization, in particular to a method for in-vitro preservation of agapanthus praecox germplasm resources and application, and the steps are as follows: after the aseptic bud is cut into single buds and inoculated in 1 / 8 modified MS culture medium and cultured for 30-35 days, the single bud of the pretreated agapanthus praecox is cut into single buds and inoculated in a germplasm preservation culture medium and cultured, the bud for germplasm preservation is inoculated in a rejuvenation pre-culture medium and cultured for 15-20 days, the bud for rejuvenation pre-culture is cut into single buds and inoculated in a rejuvenation culture medium and cultured for 30-35 days; after in-vitro preservation for 10 months, the rejuvenation pre-culture and the rejuvenation culture are carried out for a total of 45-55 days, the mortality rate and the dead seedling rate are only about 10%, after continuous proliferation culture for two generations, the proliferation coefficient is consistent with that before in-vitro preservation, and the seedling for the proliferation culture does not affect the rooting rate.

Description

TECHNICAL FIELD

[0001] The application relates to the technical field of germplasm resource protection and utilization, in particular to a method for in-vitro preservation of agapanthus praecox germplasm resources and application. BACKGROUND

[0002] Agapanthus praecox is a perennial evergreen herb of the agapanthus genus of the asparagaceae family and is originally from South Africa. Agapanthus praecox is gradually recognized by the public due to its high ornamental value and strong stress resistance. With the gradual expansion of the domestic market, the domestic breeding of excellent varieties (strains) of agapanthus praecox is carried out, so that the agapanthus praecox varieties (strains) are diversified. At present, the breeding of excellent varieties of agapanthus praecox in China is mostly carried out by hybridization or mutation in a natural state, and different varieties from the original varieties are screened out, and the breeding is emphasized rather than the selection.

[0003] Most of the existing germplasm preservation technologies use low-temperature cold storage methods, but low-temperature cold storage requires specific environment and facility equipment, and the investment and preservation cost are high. There is no specific germplasm preservation technology for agapanthus praecox, and there is no method for preserving agapanthus praecox germplasm by using biochemical reagents. If the existing tissue culture technology is used for agapanthus praecox, a large amount of manpower and material resources are needed for preservation, and on the other hand, the excessive number of subcultures will lead to variety degeneration, hormone accumulation and increased variation probability.

[0004] The existing technology has disclosed various methods for agapanthus praecox tissue culture propagation, which has a positive effect on the rapid propagation of agapanthus praecox, but the problems of variety degeneration and hormone accumulation are becoming increasingly serious. Therefore, it is of great significance to establish a long-term in-vitro preservation technology for agapanthus praecox for the preservation of agapanthus praecox germplasm resources. SUMMARY

[0005] The application aims at the problems existing in the prior art, and provides a method for in-vitro preservation of agapanthus praecox germplasm resources and application.

[0006] To achieve the above-mentioned purpose, the technical scheme adopted by the application is as follows: a method for in-vitro preservation of agapanthus praecox germplasm resources, comprising the following steps:

[0007] (1) pretreatment: taking the sterile buds of agapanthus praecox, cutting off the leaves, and cutting into single buds, and inoculating the single buds into 1 / 8 modified MS medium, and placing the medium in a temperature of 25±2 DEG C, and performing light and dark alternating culture for 30-35 days with 8 hours of light per day and light intensity of 1000-2000 lx;

[0008] (2) germplasm preservation culture: cutting off the leaves of the single buds of agapanthus praecox pretreated in the above step, and inoculating the single buds into 1 / 4 modified MS+ABA 0.2-0.3 mg / L+ sorbitol 40-50 g / L germplasm preservation medium, and placing the medium in a temperature of 25±2 DEG C, and performing light and dark alternating culture for 6 hours per day with light intensity of 1000-2000 lx.

[0009] (3) rejuvenation pre-culture: the bud of the germplasm preservation is cut to remove the aging part, inoculated in 1 / 2MS+GA 3.0-4.0mg / L rejuvenation pre-culture medium, placed in temperature 25±2℃, daily light 12h, light intensity 2000-3000lx light and dark alternating culture for 15-20d;

[0010] (4) rejuvenation culture: the bud of the rejuvenation pre-culture is cut into single bud, inoculated in MS+NAA 0.1-0.2mg / L+6-BA 0.1-0.2mg / L+GA3 0.5-0.6mg / L rejuvenation culture medium, placed in temperature 25±2℃, daily light 12h, light intensity 2000-3000lx light and dark alternating culture for 30-35d;

[0011] The improved MS is that the rest of the ingredients remain unchanged, the concentration of sucrose is reduced to 20g / L, the pH is adjusted to 6.2-6.4, and the agar concentration is increased by 1.0-1.5g / L compared with the original.

[0012] Further: the formula of the germplasm preservation medium is: 1 / 4 improved MS+ABA 0.25mg / L+ sorbitol 45g / L.

[0013] Further: the formula of the rejuvenation pre-culture is: 1 / 2MS+GA 3.5mg / L.

[0014] Further: the formula of the rejuvenation culture is: MS+NAA 0.15mg / L+6-BA 0.15mg / L+GA3 0.55mg / L.

[0015] For those skilled in the art, using the method of the application to carry out in vitro preservation of agapanthus germplasm also belongs to the protection scope of the application.

[0016] The beneficial technical effects of the application are:

[0017] 1. The application is simple to operate and can be preserved without special environment and special equipment. Generally, a unit with tissue culture laboratory only needs to adjust the medium formula, adjust the light time and the number of lamps to use the application.

[0018] 2. When agapanthus is cultured in vitro, it begins to decline after about 40 days, a small amount of yellowing appears after about 60 days, a large amount of death begins to appear after about 90 days, and almost all of them die after 5-6 months. The application does not need to be transferred within 10 months.

[0019] 3、the death rate and the dead seedling rate are only about 10% after the 10-month in-vitro preservation, the rejuvenation pre-culture and the rejuvenation culture for a total of 45-55 days, and the propagation coefficient is consistent with that before the in-vitro preservation after the proliferation culture for two generations, and the rooting rate is not affected when the seedlings after the proliferation culture are subjected to the rooting culture. BRIEF DESCRIPTION OF DRAWINGS

[0020] In order to more clearly illustrate the technical solutions in the embodiments of the present application or the prior art, the drawings needed to be used in the embodiments or the prior art description will be briefly introduced. Obviously, the drawings in the following description only constitute some embodiments of the present application, and other drawings can be obtained by those skilled in the art without creative labor.

[0021] Figure 1 is the state of the in-vitro preservation for 10 months in embodiment 2 of the present application.

[0022] Figure 2 is the state of the rejuvenation pre-culture for 15 days in embodiment 2 of the present application.

[0023] Figure 3 is the state after the rejuvenation culture for 30 days in embodiment 2 of the present application.

[0024] Figure 4 is the state after the rejuvenation culture for 35 days in embodiment 2 of the present application.

[0025] Figure 5 is the state of the in-vitro preservation for 2 months in comparative example 1 of the present application.

[0026] Figure 6 is the state of the in-vitro preservation for 4 months in comparative example 1 of the present application.

[0027] Figure 7 is the state of the in-vitro preservation for 6 months in comparative example 1 of the present application. DETAILED DESCRIPTION

[0028] The technical solutions in the embodiments of the present application will be described clearly and completely below with reference to the drawings in the embodiments of the present application. Obviously, the described embodiments only constitute some embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative labor fall within the scope of protection of the present application.

[0029] Embodiment 1

[0030] A method for in-vitro preservation of agapanthus germplasm resources, comprising the following steps:

[0031] (1) Pretreatment: take the aseptic bud of agapanthus, cut off the leaves, inoculate in 1 / 8 modified MS medium, place in temperature 25±2℃, light and dark alternation culture for 33 days with 8h light per day and light intensity 1500lx.

[0032] (2) Germplasm preservation culture: cut off the leaves of the agapanthus bud after pretreatment, inoculate in 1 / 4 modified MS+ABA 0.25mg / L+ sorbitol 45g / L germplasm preservation medium, place in temperature 25±2℃, light and dark alternation culture for 11 months with 6h light per day and light intensity 1500lx.

[0033] Among them, the modified MS is the same as the original except that the sucrose concentration is reduced to 20g / L, the pH is adjusted to 6.3, and the agar concentration is increased by 1.3g / L.

[0034] (3) Revitalization pre-culture: cut off the aged part of the bud preserved in germplasm, inoculate in 1 / 2MS+GA 3.5mg / L revitalization pre-culture medium, place in temperature 25±2℃, light and dark alternation culture for 17 days with 12h light per day and light intensity 2500lx.

[0035] (4) Revitalization culture: cut the bud after revitalization pre-culture into single bud, inoculate in MS+NAA 0.15mg / L+6-BA 0.15mg / L+GA3 0.55mg / L revitalization culture medium, place in temperature 25±2℃, light and dark alternation culture for 33 days with 12h light per day and light intensity 2500lx.

[0036] (5) Proliferation culture: cut the bud after revitalization culture into single bud after removing the leaves, inoculate in MS+IAA 0.05mg / L+ZT 1.5mg / L+BR 0.03mg / L proliferation culture medium, place in temperature 25±2℃, light and dark alternation culture for 35 days with 12h light per day and light intensity 2500lx.

[0037] (6) Repeat step (5) once, and the proliferation coefficient is 6.4.

[0038] (7) Rooting culture: cut the bud after step (6) into single bud after removing the leaves, inoculate in 1 / 2MS+NAA 0.3mg / L+IAA 0.8mg / L+coconut juice 50ml / L rooting culture medium, and the rooting rate is 98.3%.

[0039] Example 2

[0040] A method for in vitro preservation of agapanthus germplasm resources, comprising the following steps:

[0041] (1) Pretreatment: take the aloe vera sterile bud, cut off the leaves, inoculate in 1 / 8 modified MS medium, place in temperature 25±2℃, daily light 8h, light intensity 1000lx, light and dark alternation culture for 30d.

[0042] (2) Germplasm preservation culture: cut off the leaves of the aloe vera single bud after pretreatment, inoculate in 1 / 4 modified MS+ABA 0.2mg / L+ sorbitol 40g / L germplasm preservation medium, place in temperature 25±2℃, daily light 6h, light intensity 1000lx, light and dark alternation culture for 10 months, and the preservation culture result is shown in Figure 1

[0043] Among them, the modified MS is that the sucrose concentration is reduced to 20g / L, the pH is adjusted to 6.3, and the agar concentration is increased by 1.3g / L compared with the original.

[0044] (3) Revitalization pre-culture: cut off the aged part of the germplasm preservation bud, inoculate in 1 / 2MS+GA 3.0mg / L revitalization pre-culture medium, place in temperature 25±2℃, daily light 12h, light intensity 2000lx, light and dark alternation culture for 15d, and the culture result is shown in Figure 2

[0045] (4) Revitalization culture: cut the revitalization pre-culture bud into single bud, inoculate in MS+NAA 0.1mg / L+6-BA 0.1mg / L+GA3 0.5mg / L revitalization culture medium, place in temperature 25±2℃, daily light 12h, light intensity 2000lx, light and dark alternation culture for 30d, and the revitalization culture 30d state is shown in Figure 3

[0046] (5) Proliferation culture: cut the revitalization culture bud into single bud after removing the leaves, inoculate in MS+IAA 0.05mg / L+ZT1.5mg / L+BR 0.03mg / L proliferation culture medium, place in temperature 25±2℃, daily light 12h, light intensity 2500lx, light and dark alternation culture for 35d, and the proliferation culture result is shown in Figure 4

[0047] (6) Repeat step (5) twice, and the proliferation coefficient is 7.1.

[0048] (7) Rooting culture: cut the bud into single bud after removing the leaves, inoculate in 1 / 2MS+NAA 0.3mg / L+IAA 0.8mg / L+coconut milk 50ml / L rooting culture medium, and the rooting rate is 99.2%.

[0049] Example 3

[0050] A method for in vitro preservation of aloe vera germplasm resources, comprising the steps of:​​​​

[0051] (1) Pretreatment: Take the aseptic bud of agapanthus, cut off the leaves, and inoculate it in 1 / 8 modified MS medium, and place it in a temperature of 25±2℃, with 8h light per day, light intensity of 2000lx, and light and dark alternation for 35d.

[0052] (2) Germplasm preservation culture: cut off the leaves of the agapanthus single bud after pretreatment, inoculate it in 1 / 4 modified MS + ABA 0.3mg / L + sorbitol 50g / L germplasm preservation medium, and place it in a temperature of 25±2℃, with 6h light per day, light intensity of 2000lx, and light and dark alternation for 9 months.

[0053] Among them, the modified MS is unchanged in other components, the sucrose concentration is reduced to 20g / L, the pH is adjusted to 6.3, and the agar concentration is increased by 1.3g / L compared with the original.

[0054] (3) Revitalization pre-culture: cut off the aged part of the germplasm preservation bud, inoculate it in 1 / 2MS + GA 4.0mg / L revitalization pre-culture medium, and place it in a temperature of 25±2℃, with 12h light per day, light intensity of 3000lx, and light and dark alternation for 20d.

[0055] (4) Revitalization culture: cut the revitalization pre-cultured bud into single buds, inoculate it in MS + NAA 0.2mg / L + 6-BA 0.2mg / L + GA3 0.6mg / L revitalization culture medium, and place it in a temperature of 25±2℃, with 12h light per day, light intensity of 3000lx, and light and dark alternation for 35d.

[0056] (5) Proliferation culture: cut the revitalization cultured bud into single buds after removing the leaves, inoculate it in MS + IAA 0.05mg / L + ZT 1.5mg / L + BR 0.03mg / L proliferation culture medium, and place it in a temperature of 25±2℃, with 12h light per day, light intensity of 2500lx, and light and dark alternation for 35d.

[0057] (6) Repeat step (5) twice, and the proliferation coefficient is 7.2.

[0058] (7) Rooting culture: cut the bud into single buds after removing the leaves, inoculate it in 1 / 2MS + NAA 0.3mg / L + IAA 0.8mg / L + coconut juice 50ml / L rooting culture medium, and the rooting rate is 98.9%.

[0059] Comparative Example 1

[0060] According to the patent "Agapanthus Tissue Culture and Rapid Propagation Method" (publication number: CN116649221A), primary culture and proliferation culture were carried out, and the revitalization pre-culture and revitalization culture were carried out according to Example 1 of the present application after being stored for 2 months, 4 months and 6 months during the proliferation culture. The results showed that after being stored for 2 months (such asFigure 5 As shown in Table 2, about 11.8% of the seedlings died, the non-dead seedlings were rejuvenated, the rejuvenation rate was 90.4% at the time of rejuvenation, the seedlings were cultured for 3 generations after rejuvenation, the proliferation coefficient was 6.6, the seedlings were cultured for rooting after proliferation, and the rooting rate was 100%; after being stored for 4 months (as shown in Table 2), about 76.9% of the seedlings died, the non-dead seedlings were rejuvenated, the rejuvenation rate was only 42.1% at the time of rejuvenation, about 46.7% of the seedlings became dead seedlings after being cultured for 3 generations after rejuvenation, the proliferation coefficient was 3.1, the seedlings were cultured for rooting after proliferation, and the rooting rate was 84.3%; after being stored for 6 months (as shown in Table 2), almost all of the seedlings died, the non-dead seedlings were rejuvenated, the rejuvenation rate was only 3.6% at the time of rejuvenation, about 90.1% of the seedlings became dead seedlings after being cultured for 3 generations after rejuvenation, the proliferation coefficient was 1.9, and the rooting rate was 46.0% after the seedlings were cultured for rooting. Figure 6 Figure 7

[0061] Finally, it should be noted that the above examples are only used to illustrate but not limit the technical solutions of the present application. Although the present application has been described in detail with reference to the above examples, those skilled in the art should understand that the present application can still be modified or replaced equivalently without departing from the spirit and scope of the present application, and any modification or partial replacement should be covered in the scope of the claims of the present application.​​

Claims

1. A method for in vitro preservation of Agapanthus germplasm resources, characterized by the following steps: include: (1) Pretreatment: Take sterile buds of Agapanthus, remove the leaves and cut them into single buds, inoculate them in 1 / 8 modified MS medium, place them at a temperature of 25±2℃, irradiate with light for 8 hours a day, and culture them in alternating light and dark for 30 to 35 days with a light intensity of 1000 to 2000 lx. (2) Germplasm preservation culture: The leaves of the pretreated Agapanthus buds were cut off and inoculated into 1 / 4 of the modified MS + ABA 0.2-0.3 mg / L + sorbitol 40-50 g / L germplasm preservation culture medium. The medium was placed at 25±2℃, with light for 6 hours a day and light intensity of 1000-2000 lx for alternating light and dark culture. (3) Rejuvenation pre-culture: The germplasm-preserved buds were inoculated in 1 / 2 MS + GA 3.0~4.0 mg / L rejuvenation pre-culture medium, placed at a temperature of 25±2℃, and irradiated with light for 12 hours a day with a light intensity of 2000~3000 lx for 15~20 days of alternating light and dark culture. (4) Rejuvenation culture: The buds that have undergone rejuvenation culture are cut into single buds and inoculated into MS+NAA 0.1~0.2mg / L+6-BA 0.1~0.2mg / L+GA3 0.5~0.6mg / L rejuvenation culture medium. The buds are placed at a temperature of 25±2℃, and are exposed to light for 12 hours a day with a light intensity of 2000~3000lx. The buds are cultured in alternating light and dark conditions for 30~35 days. The modified MS has the other components unchanged, but the sucrose concentration is reduced to 20 g / L, the pH is adjusted to 6.2-6.4, and the agar concentration is increased by 1.0-1.5 g / L compared to the original.

2. The method according to claim 1, characterized in that: The formula for the germplasm preservation medium is: 1 / 4 modified MS + ABA 0.25 mg / L + sorbitol 45 g / L.

3. The method according to claim 1, characterized in that: The formulation for the rejuvenation pre-culture was: 1 / 2 MS + GA 3.5 mg / L.

4. The method according to claim 1, characterized in that: The formulation for the rejuvenation culture is: MS + NAA 0.15 mg / L + 6-BA 0.15 mg / L + GA3 0.55 mg / L.

5. The application of the method according to any one of claims 1-4 in the in vitro preservation of Agapanthus germplasm.

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