African agapanthus germplasm in-vitro preservation method and application

Abstract

The invention relates to the technical field of germplasm resource protection and utilization, in particular to an agapanthus germplasm resource in-vitro preservation method and application, and the method comprises the following steps: cutting off leaves of sterile buds, cutting into single buds, inoculating into a 1 / 8 improved MS culture medium, culturing for 30-35 days, cutting off leaves of the pretreated agapanthus single buds, inoculating into a germplasm preservation culture medium, and culturing for 30-35 days; inoculating germplasm preserved buds into a rejuvenation pre-culture medium for culturing for 15-20 days, cutting the rejuvenation pre-cultured buds into single buds, and inoculating the single buds into a rejuvenation culture medium for culturing for 30-35 days; after in-vitro preservation for 10 months, rejuvenation pre-culture and rejuvenation culture are carried out for 45-55 days in total, the death rate and the dead seedling rate are only about 10%, after continuous 2 generations of multiplication culture, the multiplication coefficient is consistent with that before in-vitro preservation, and meanwhile the rooting rate of the seedlings subjected to multiplication culture is not affected when rooting culture is carried out.

Description

Technical Field

[0001] The invention relates to the technical field of germplasm resource protection and utilization, and in particular to an in vitro preservation method and application of Agapanthus germplasm resources. Background Art

[0002] Agapanthus praecox is a perennial evergreen herbaceous plant of the Amaryllis family. It is native to South Africa and has gradually become known to the public due to its high ornamental value and strong resistance to adversity. With the gradual expansion of the domestic market, the selection and breeding of excellent varieties (lines) of Agapanthus praecox has been gradually carried out in China, making the varieties (lines) of Agapanthus praecox diversified. However, at present, the selection and breeding of excellent varieties of Agapanthus praecox in China is mostly through hybridization or mutation under natural conditions, and the method of selecting varieties different from the original ones focuses on selection rather than breeding.

[0003] Most plants with existing germplasm preservation technology are preserved by low-temperature refrigeration, but low-temperature refrigeration requires a specific environment and facilities and equipment, with large investments and high preservation costs. For Agapanthus, there is no specific germplasm preservation technology, nor is there a method to preserve Agapanthus germplasm through biochemical reagents. If Agapanthus is continuously subcultured using existing tissue culture technology, a large amount of manpower and material resources will be required for preservation. On the other hand, too many subcultures will lead to variety degeneration, hormone accumulation, and an increased probability of mutation.

[0004] The prior art has disclosed a variety of methods for propagating Agapanthus by tissue culture, which has played a positive role in the rapid propagation of Agapanthus, but the problems of variety degeneration and hormone accumulation are becoming increasingly serious. Therefore, establishing a long-term in vitro preservation technology for Agapanthus is of great significance to the preservation of Agapanthus germplasm resources. Summary of the invention

[0005] The purpose of the present invention is to provide a method and application for in vitro preservation of Agapanthus germplasm resources in view of the problems existing in the prior art.

[0006] To achieve the above object, the technical solution adopted by the present invention is: a method for in vitro preservation of Agapanthus germplasm resources, comprising the following steps:

[0007] (1) Pretreatment: Take sterile buds of Agapanthus nymphaeaceae, remove the leaves and cut into single buds, inoculate them in 1 / 8 modified MS medium, place at 25±2℃, light for 8 hours per day, light intensity of 1000-2000lx, and culture them in alternating light and dark for 30-35 days;

[0008] (2) Germplasm preservation culture: The leaves of the single buds of Agapanthus nymphaeaceae that had been pre-treated were cut off and inoculated into a germplasm preservation medium containing 1 / 4 modified MS + ABA 0.2-0.3 mg / L + sorbitol 40-50 g / L. The medium was placed at a temperature of 25±2°C, with light exposure for 6 hours per day and a light intensity of 1000-2000 lx under alternating light and dark conditions;

[0009] (3) Rejuvenation pre-culture: The senescent part of the germplasm-preserved buds is cut off and inoculated into a rejuvenation pre-culture medium containing 1 / 2MS + GA 3.0-4.0 mg / L. The culture medium is placed at 25±2°C, with light exposure for 12 hours per day and a light intensity of 2000-3000 lx for 15-20 days in alternating light and dark conditions.

[0010] (4) Rejuvenation culture: Cut the buds from the rejuvenation pre-culture into single buds and inoculate them into a rejuvenation medium containing MS + NAA 0.1-0.2 mg / L + 6-BA 0.1-0.2 mg / L + GA3 0.5-0.6 mg / L. Place the medium at 25±2°C, light for 12 hours per day, and light intensity of 2000-3000 lx for 30-35 days in alternating light and dark conditions.

[0011] The improved MS has the following characteristics: the other components remain unchanged, 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 with the original one.

[0012] Furthermore: the formula of the germplasm preservation medium is: 1 / 4 modified 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+GA30.55mg / L.

[0015] For those skilled in the art, using the method of the present invention to preserve Agapanthus germplasm in vitro also falls within the protection scope of this application.

[0016] The beneficial technical effects of the present invention are:

[0017] 1. The present invention is easy to operate and can be preserved without special environment or special equipment. Generally, units with tissue culture laboratories only need to adjust the culture medium formula, the illumination time, and the number of lamps to use the present invention.

[0018] 2. When Agapanthus is cultured in vitro, it begins to decline after about 40 days, becomes slightly withered and yellow after about 60 days, and begins to die in large numbers after about 90 days. Almost all of them die after 5 to 6 months. However, the present invention does not transfer within 10 months.

[0019] 3. After 10 months of in vitro storage, the present invention performs rejuvenation pre-culture and rejuvenation culture for a total of 45 to 55 days, and the mortality rate and dead seedling rate are only about 10%. After two consecutive generations of proliferation culture, the proliferation coefficient is consistent with that before in vitro storage. At the same time, when the seedlings undergoing proliferation culture are subjected to rooting culture, their rooting rate is not affected. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative labor.

[0021] Figure 1 This is the state of in vitro storage for 10 months in Example 2 of the present invention.

[0022] Figure 2 This is the state of 15 days of rejuvenation pre-culture in Example 2 of the present invention.

[0023] Figure 3 This is the state of Example 2 of the present invention after 30 days of rejuvenation culture.

[0024] Figure 4 This is the state of proliferation culture 35 days after rejuvenation culture in Example 2 of the present invention.

[0025] Figure 5 This is the state of comparative example 1 of the present invention stored in vitro for 2 months.

[0026] Figure 6 This is the state of comparative example 1 of the present invention after being stored in vitro for 4 months.

[0027] Figure 7 This is the state of comparative example 1 of the present invention after being stored in vitro for 6 months. DETAILED DESCRIPTION

[0028] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0029] Example 1

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

[0031] (1) Pretreatment: Take sterile buds of Agapanthus, remove the leaves and cut into single buds, inoculate them in 1 / 8 modified MS medium, place at 25±2℃, light for 8 hours per day, light intensity of 1500lx, and culture for 33 days in alternating light and dark.

[0032] (2) Germplasm preservation culture: Cut off the leaves of the single bud of Agapanthus that has been pre-treated, inoculate it into 1 / 4 modified MS + ABA 0.25 mg / L + sorbitol 45 g / L germplasm preservation medium, place it at a temperature of 25 ± 2 ° C, light for 6 hours per day, and light intensity of 1500 lx for 11 months in a light-dark alternating culture system.

[0033] Among them, the improved MS has the remaining ingredients unchanged, 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.

[0034] (3) Rejuvenation pre-culture: Cut off the senescent part of the germplasm-preserved buds, inoculate them into a rejuvenation pre-culture medium of 1 / 2MS + GA 3.5 mg / L, place them at a temperature of 25±2℃, light for 12 hours per day, and light intensity of 2500lx for 17 days of alternating light and dark.

[0035] (4) Rejuvenation culture: Cut the buds pre-cultured for rejuvenation into single buds, inoculate them into rejuvenation medium consisting of MS + NAA 0.15 mg / L + 6-BA 0.15 mg / L + GA3 0.55 mg / L, place them at a temperature of 25±2℃, light for 12 hours per day, and light intensity of 2500 lx for 33 days of alternating light and dark.

[0036] (5) Proliferation culture: Remove the leaves of the buds that have undergone rejuvenation culture and cut them into single buds. Inoculate them into a proliferation medium consisting of MS + IAA 0.05 mg / L + ZT 1.5 mg / L + BR 0.03 mg / L, place them at a temperature of 25 ± 2 °C, illuminate them for 12 h per day, and culture them in a light-dark alternating system with a light intensity of 2500 lx for 35 days.

[0037] (6) Repeat step (5) once, with a proliferation factor of 6.4.

[0038] (7) Rooting culture: The buds cultured in step (6) were defoliated and cut into single buds, which were inoculated into a rooting medium consisting of 1 / 2MS + 0.3 mg / L NAA + 0.8 mg / L IAA + 50 ml / L coconut juice. The rooting rate was 98.3%.

[0039] Example 2

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

[0041] (1) Pretreatment: Take sterile buds of Agapanthus, remove the leaves and cut into single buds, inoculate them in 1 / 8 modified MS medium, place at 25±2℃, light for 8 hours per day, light intensity of 1000lx, and culture for 30 days in alternating light and dark.

[0042] (2) Germplasm preservation culture: Cut off the leaves of the pre-treated single bud of Agapanthus nymphaeaceae, inoculate it in 1 / 4 modified MS + ABA 0.2 mg / L + sorbitol 40 g / L germplasm preservation medium, place it at a temperature of 25 ± 2 ° C, light for 6 hours per day, light intensity of 1000 lx, and culture it in alternating light and dark for 10 months. The preservation culture results are as follows: Figure 1 shown.

[0043] Among them, the improved MS has the remaining ingredients unchanged, 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) Rejuvenation pre-culture: Cut off the senescent part of the germplasm-preserved buds, inoculate them into a rejuvenation pre-culture medium containing 1 / 2MS + GA 3.0 mg / L, place them at a temperature of 25±2℃, light for 12 hours per day, and light intensity of 2000lx for 15 days of alternating light and dark. The culture results are as follows: Figure 2 shown.

[0045] (4) Rejuvenation culture: Cut the buds from the rejuvenation pre-culture into single buds and inoculate them into a rejuvenation medium containing MS + NAA 0.1 mg / L + 6-BA 0.1 mg / L + GA3 0.5 mg / L. Place the buds at 25 ± 2 °C, light for 12 h per day, and light intensity of 2000 lx for 30 days of alternating light and dark. The state of the buds after 30 days of rejuvenation culture is as follows: Figure 3 shown.

[0046] (5) Proliferation culture: Remove the leaves of the buds that have undergone rejuvenation culture and cut them into single buds. Inoculate them in a proliferation medium containing MS + IAA 0.05 mg / L + ZT 1.5 mg / L + BR 0.03 mg / L. Place them at a temperature of 25 ± 2 °C, illuminate them for 12 h per day, and culture them in a light-dark alternating system with a light intensity of 2500 lx for 35 days. The proliferation culture results are as follows: Figure 4 shown.

[0047] (6) Repeat step (5) twice, with a proliferation factor of 7.1.

[0048] (7) Rooting culture: The buds cultured in step (6) were defoliated and cut into single buds, which were inoculated into a rooting medium consisting of 1 / 2MS + 0.3 mg / L NAA + 0.8 mg / L IAA + 50 ml / L coconut juice. The rooting rate was 99.2%.

[0049] Example 3

[0050] A method for in vitro preservation of Agapanthus germplasm resources, comprising the following steps:

[0051] (1) Pretreatment: Take sterile buds of Agapanthus, remove the leaves and cut into single buds, inoculate them in 1 / 8 modified MS medium, place at 25±2℃, light for 8 hours per day, light intensity of 2000lx, and culture for 35 days in alternating light and dark.

[0052] (2) Germplasm preservation culture: Cut off the leaves of the single bud of Agapanthus that has been pre-treated, inoculate it into 1 / 4 modified MS + ABA 0.3 mg / L + sorbitol 50 g / L germplasm preservation medium, place it at a temperature of 25 ± 2 ° C, light for 6 hours per day, and light intensity of 2000 lx for 9 months in alternating light and dark.

[0053] Among them, the improved MS has the remaining ingredients unchanged, 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) Rejuvenation pre-culture: Cut off the senescent part of the germplasm-preserved buds, inoculate them into 1 / 2MS + GA 4.0 mg / L rejuvenation pre-culture medium, place them at 25±2℃, light for 12 hours per day, light intensity of 3000lx, and culture them in alternating light and dark for 20 days.

[0055] (4) Rejuvenation culture: Cut the buds pre-cultured for rejuvenation into single buds, inoculate them into rejuvenation medium consisting of MS + NAA 0.2 mg / L + 6-BA 0.2 mg / L + GA3 0.6 mg / L, place them at a temperature of 25 ± 2 °C, light for 12 h per day, and culture them in a light intensity of 3000 lx with alternating light and dark for 35 days.

[0056] (5) Proliferation culture: Remove the leaves of the buds that have undergone rejuvenation culture and cut them into single buds. Inoculate them into a proliferation medium consisting of MS + IAA 0.05 mg / L + ZT 1.5 mg / L + BR 0.03 mg / L, place them at a temperature of 25 ± 2 °C, illuminate them for 12 h per day, and culture them in a light-dark alternating system with a light intensity of 2500 lx for 35 days.

[0057] (6) Repeat step (5) twice, with a proliferation coefficient of 7.2.

[0058] (7) Rooting culture: The buds cultured in step (6) were defoliated and cut into single buds, which were inoculated into a rooting medium consisting of 1 / 2MS + 0.3 mg / L NAA + 0.8 mg / L IAA + 50 ml / L coconut juice. The rooting rate was 98.9%.

[0059] Comparative Example 1

[0060] According to the patent "Agapanthus tissue culture rapid propagation method" (publication number: CN116649221A), primary and proliferation cultures were carried out, wherein the proliferation culture was stored for 2 months, 4 months, and 6 months, and then the rejuvenation pre-culture and rejuvenation culture were carried out according to Example 1 of the present invention. The results showed that after 2 months of storage (such as Figure 5 As shown in the figure, about 11.8% of the seedlings have died. The seedlings that did not die were rejuvenated, and the rejuvenation rate was 90.4%. After rejuvenation, they were multiplied for 3 generations, with a proliferation coefficient of 6.6. After multiplication, they were rooted and the rooting rate was 100%. After 4 months of storage (as shown in the figure), about 11.8% of the seedlings died. The seedlings that did not die were rejuvenated, with a rejuvenation rate of 90.4%. After rejuvenation, they were multiplied for 3 generations, with a proliferation coefficient of 6.6. After multiplication, they were rooted and the rooting rate was 100%. Figure 6 As shown in the figure, about 76.9% of the seedlings died. The seedlings that did not die were rejuvenated, and the rejuvenation rate was only 42.1%. After rejuvenation, about 46.7% of the seedlings were recultured for 3 generations, and the proliferation coefficient was 3.1. After proliferation, rooting culture was carried out, and the rooting rate was 84.3%. After 6 months of storage (as shown in the figure), about 76.9% of the seedlings died. The seedlings that did not die were recultured, and the rejuvenation rate was only 42.1%. After 3 generations of proliferation culture, about 46.7% of the seedlings were recultured for 3 generations, and the proliferation coefficient was 3.1. After 3 generations of proliferation, rooting culture was carried out, and the rooting rate was 84.3%. Figure 7 Almost all the seedlings (as shown) died. For the seedlings that did not obviously die, the rejuvenation rate was only 3.6%. After rejuvenation, about 90.1% of them became stiff seedlings after three generations of proliferation culture, with a proliferation coefficient of 1.9. After proliferation, they were rooted and the rooting rate was 46.0%.

[0061] Finally, it should be noted that the above embodiments are only used to illustrate rather than limit the technical solutions of the present invention. Although the present invention has been described in detail with reference to the above embodiments, those skilled in the art should understand that the present invention can still be modified or replaced by equivalents. Any modification or partial replacement that does not depart from the spirit and scope of the present invention should be included in the scope of the claims of the present invention.

Claims

1. A method for in vitro preservation of Agapanthus germplasm resources, characterized in that the steps include: (1) Pretreatment: Take sterile buds of Agapanthus nymphaeaceae, remove the leaves and cut into single buds, inoculate them in 1 / 8 modified MS medium, place at 25±2℃, light for 8 hours per day, light intensity of 1000-2000lx, and culture them in alternating light and dark for 30-35 days; (2) Germplasm preservation culture: The leaves of the single buds of Agapanthus nymphaeaceae that had been pre-treated were cut off and inoculated into a germplasm preservation medium containing 1 / 4 modified MS + ABA 0.2-0.3 mg / L + sorbitol 40-50 g / L. The medium was placed at a temperature of 25±2°C, with light exposure for 6 hours per day and a light intensity of 1000-2000 lx under alternating light and dark conditions; (3) Rejuvenation pre-culture: The germplasm-preserved buds are inoculated in a rejuvenation pre-culture medium containing 1 / 2MS + GA 3.0-4.0 mg / L, placed at a temperature of 25±2°C, illuminated for 12 hours per day, and cultured in a light-dark alternating system with a light intensity of 2000-3000 lx for 15-20 days; (4) Rejuvenation culture: Cut the buds from the rejuvenation pre-culture into single buds and inoculate them into a rejuvenation medium containing MS + NAA 0.1-0.2 mg / L + 6-BA 0.1-0.2 mg / L + GA3 0.5-0.6 mg / L. Place the medium at 25±2°C, light for 12 hours per day, and light intensity of 2000-3000 lx for 30-35 days in alternating light and dark conditions. The improved MS has the following characteristics: the other components remain unchanged, 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 with the original one.

2. The method according to claim 1, characterized in that: The formula of 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 formula of the rejuvenation pre-culture is: 1 / 2MS+GA 3.5mg / L.

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

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

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