Clonal date palm germplasm resource in-vitro preservation method and application

Abstract

The invention provides a clone date palm germplasm resource in-vitro preservation method and application. Belongs to the technical field of germplasm resource preservation. According to the method, calluses or test-tube plantlets are used as preservation objects, and a specific in-vitro preservation culture medium and preservation conditions are determined. The method effectively ensures the genetic integrity and stability of genetic materials of the date palm, the callus of the date palm can be effectively preserved for 150-180 days, and within the time range, the browning rate of the callus is controlled to be 10% or below, the vitrification rate is controlled to be 9% or below, and the primary differentiation rate can reach 67%; the test-tube plantlet of the date palm can be effectively preserved for 8-10 months, and in the time range, leaves of the test-tube plantlet can be prevented from chlorosis and withering. After the preservation is finished, the growth can be recovered and the root system can be induced after 20 days of subculture, and the survival rate can reach 80%.

Description

Technical Field

[0001] The present invention relates to the technical field of germplasm resource preservation, and more specifically, to a method and application for in vitro preservation of clonal date palm germplasm resources. Background Art

[0002] Date palm trees are perennial tropical palm plants, dioecious, and are important woody food crops in Arab countries. Arab countries have rich date palm resources. In recent years, China has introduced high-quality date palm resources from the United Arab Emirates, Egypt, Pakistan and other countries through international cooperation projects, filling the gap in the lack of maternal date palm resources in China. The germplasm resources of date palm are an important material basis for breeding and genetic research, requiring the genetic integrity and stability of the genetic material contained in date palm individuals to be maintained within a certain time and space, and ensuring that its genetic characteristics can be transmitted to the next generation through different reproductive methods.

[0003] Date palms can reproduce offspring by seeds, but because they are dioecious plants and are in a highly heterozygous state, this reproductive method will lose the target traits of the parents in production and cannot well guarantee the excellent characteristics and fruit characteristics of the mother plant. Therefore, it is impossible to achieve the preservation of high-quality date palm resources by preserving seeds. Due to the special reproductive method of date palms, how to achieve the in vitro preservation of excellent germplasm resources has become the top priority in the development of the date palm industry.

[0004] At present, date palm trees are mainly preserved in the form of tiller seedlings internationally. However, due to the large size of the tree body, tiller seedlings can only be planted and preserved in the field, and it requires a large amount of manpower, material resources and financial resources, and is easily affected by drought, waterlogging and pests and diseases. The in vitro preservation time at normal temperature is short, and more frequent subculture is required, increasing the labor cost and also increasing the probability of clonal variation. In vitro preservation of date palm resources can be achieved at low cost and high efficiency through plant tissue culture, which is undoubtedly the best preservation method at present.

[0005] However, at the present stage, there are few records on the in vitro preservation of clonal date palm germplasm resources at home and abroad.

[0006] Therefore, how to provide a method and application for in vitro preservation of clonal date palm germplasm resources that can effectively retain the genetic integrity and stability of date palm genetic material is a technical problem that needs to be solved urgently by those skilled in the art. Summary of the Invention

[0007] In view of this, the present invention provides a method for in vitro preservation of clonal date palm germplasm resources, which effectively guarantees the genetic integrity and stability of date palm genetic material. The callus of date palm can be effectively preserved for 150 - 180 days, and the differentiation rate of primary culture after the preservation is 67%; the test-tube seedlings of date palm can be effectively preserved for 8 - 10 months. After the preservation, they can resume growth after 20 days of subculture, induce roots, and the survival rate can reach 80%.

[0008] To achieve the above object, the present invention adopts the following technical solutions:

[0009] A method for in vitro preservation of clonal date palm germplasm resources includes the following steps:

[0010] (1) Preparation of date palm germplasm resources: After selecting the tissue of date palm for sterilization, it is placed in a culture medium for culture to obtain the callus or test-tube plantlets to be preserved.

[0011] (2) Preparation of in vitro preservation culture medium: The callus preservation culture medium is as follows: MS + 30 g / L sucrose + 6 mg / L uniconazole + 1 mg / L 6-BA + 1 mg / L 2,4-D + 3.5 g / L phytagel + 20 g / L mannitol + 0.5 g / L activated carbon; The test-tube plantlet preservation culture medium is as follows: 1 / 2MS + 90 g / L sucrose + 30 g / L mannitol + 0.5 mg / L NAA + 3 mg / L chlormequat chloride + 0.5 mg / L paclobutrazol.

[0012] (3) In vitro preservation: The callus described in step (1) is placed in the callus preservation culture medium described in step (2) for in vitro preservation;

[0013] Or the test-tube plantlets described in step (1) are placed in the test-tube plantlet preservation culture medium described in step (2) for in vitro preservation.

[0014] Preferably, the preparation process of the callus described in step (1) is as follows: Select the shoot apical meristem of date palm tissue as the explant, and after sterilization, carry out tissue culture to obtain the callus to be preserved.

[0015] Preferably, the preparation process of the test-tube plantlets described in step (1) is as follows: Select the shoot apical growth tissue of date palm as the explant, obtain callus through tissue culture, induce the callus to form embryogenic callus, the embryogenic callus differentiates into date palm somatic embryos, and the somatic embryos germinate into test-tube plantlets.

[0016] Preferably, the in vitro preservation conditions of the callus described in step (3) are as follows: Dark culture preservation at 10 - 15 °C.

[0017] Preferably, the in vitro preservation conditions of the test-tube plantlets described in step (3) are as follows: Culture and preservation under weak light conditions of 500 - 1000 Lux at 10 - 15 °C.

[0018] Preferably, the preservation time of the callus described in step (3) is 150 - 180 d; the preservation time of the test-tube plantlets is 8 - 10 months.

[0019] Another object of the present invention is to provide: The application of the above-mentioned method for in vitro preservation of clonal date palm germplasm resources in the in vitro preservation of clonal date palm germplasm resources.

[0020] Another object of the present invention is to provide: an in vitro conservation medium for clonal date palm germplasm resources, which medium includes a callus conservation medium and / or a test-tube plantlet conservation medium. The callus conservation medium is as follows: MS + 30 g / L sucrose + 6 mg / L uniconazole + 1 mg / L 6-BA + 1 mg / L 2,4-D + 3.5 g / L phytagel + 20 g / L mannitol + 0.5 g / L activated carbon; the test-tube plantlet conservation medium is as follows: 1 / 2 MS + 90 g / L sucrose + 30 g / L mannitol + 0.5 mg / L NAA + 3 mg / L chlormequat chloride + 0.5 mg / L paclobutrazol.

[0021] Another object of the present invention is to provide: the application of the above in vitro conservation medium for clonal date palm germplasm resources in the in vitro conservation of clonal date palm germplasm resources.

[0022] As can be seen from the above technical solutions, compared with the prior art, the present invention has the following beneficial effects:

[0023] The present invention provides a method for in vitro conservation of clonal date palm germplasm resources, using callus or test-tube plantlets as the conservation objects, and determining specific in vitro conservation media and conservation conditions. It effectively ensures the genetic integrity and stability of the genetic material of date palm. The callus of date palm can be effectively conserved for 150 - 180 days. Within this time range, the browning rate of the callus is controlled below 10%, the vitrification is controlled below 9%, and the primary differentiation rate can reach 67%; the test-tube plantlets of date palm can be effectively conserved for 8 - 10 months. Within this time range, the test-tube plantlets can maintain their leaves without chlorosis and without withering. After the conservation ends, subculture for 20 days can restore growth and induce roots, and the survival rate can reach 80%. Detailed implementation mode

[0024] The following will combine the embodiments of the present invention to clearly and completely describe the technical solutions of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts shall fall within the protection scope of the present invention.

[0025] Example 1

[0026] A method for in vitro conservation of clonal date palm germplasm resources includes the following steps

[0027] (1) Preparation of date palm germplasm resources: Select the shoot apical meristem of adult date palm trees as explants. Immerse the explants in 75% alcohol for 30 s, rinse them thoroughly with sterile water, and then soak them in 2% sodium hypochlorite for 15 min for disinfection. After disinfection, place them in a medium of MS + 3.5 g / L phytagel + 30 g / L sucrose + 1 g / L activated carbon + 150 mg / L glutamine + 0.3 mg / L nicotinic acid + 0.5 mg / L pyridoxine hydrochloride + 100 mg / L inositol + 0.7 g / L potassium dihydrogen phosphate + 2 mg / L 6-BA + 3 mg / L NAA + 5 mg / L 2,4-D

[0028] + 1 mg / L 2-ip (N6-isopentenyladenine) and culture for 30 d to obtain the callus to be preserved;

[0029] (2) Preparation of in vitro conservation medium: Callus conservation medium, MS + 30 g / L sucrose + 6 mg / L uniconazole + 1 mg / L 6-BA + 1 mg / L 2,4-D + 3.5 g / L phytagel + 20 g / L mannitol + 0.5 g / L activated carbon;

[0030] (3) In vitro conservation: Place the callus obtained in step (1) in the callus conservation medium described in step (2) and culture it in the dark at 10 - 15 °C. After 150 d of conservation, the browning rate reaches 8% and the vitrified callus reaches 7%;

[0031] (4) Recovery of callus proliferation ability: After the conservation, transfer the callus to a medium of MS + 3.5 g / L phytagel + 30 g / L sucrose + 1 g / L activated carbon + 8 mg / L 2,4-D + 3 mg / L 2-ip for proliferation culture for 30 d, and the primary recovery proliferation coefficient can reach 5.6;

[0032] (5) Recovery of callus differentiation ability: After the conservation, transfer the callus to a medium of MS + 30 g / L sucrose + 3.5 g / L phytagel + 1.5 g / L activated carbon + 45 mg / L inositol + 200 mg / L glutamine + 500 mg / L hydrolyzed casein + 0.5 mg / L 2,4-D + 0.3 mg / L 6-BA + 2 mg / L KT + 1 mg / L NAA for callus differentiation culture, and subculture every 30 d. After 3 subcultures, 67% of the callus has the ability to differentiate.

[0033] Example 2

[0034] A method for in vitro conservation of clonal date palm germplasm resources, comprising the following steps

[0035] (1) Preparation of date palm germplasm resources: Select the shoot apical meristem of adult date palm trees as explants. Immerse the explants in 75% alcohol for 30 s, rinse them thoroughly with sterile water, and then soak them in 2% sodium hypochlorite for 15 min for disinfection. After disinfection, place them in an MS + 3.5 g / L phytagel + 30 g / L sucrose + 1 g / L activated carbon + 150 mg / L glutamine + 0.3 mg / L nicotinic acid + 0.5 mg / L pyridoxine hydrochloride + 100 mg / L inositol + 0.7 g / L potassium dihydrogen phosphate + 2 mg / L 6-BA + 3 mg / L NAA + 5 mg / L 2,4-D + 1 mg / L 2-ip medium and culture for 30 d to obtain the callus to be preserved;

[0036] (2) Preparation of in vitro conservation medium: Callus conservation medium, MS + 30 g / L sucrose + 6 mg / L uniconazole + 1 mg / L 6-BA + 1 mg / L 2,4-D + 3.5 g / L phytagel + 20 g / L mannitol + 0.5 g / L activated carbon;

[0037] (3) In vitro conservation: Place the callus obtained in step (1) in the callus conservation medium described in step (2) and conduct dark culture at 10 °C. After being preserved for 180 d, the browning rate is 10%, and the vitrified callus reaches 9%;

[0038] (4) Recovery of callus proliferation ability: After the preservation is completed, transfer the callus to an MS + 3.5 g / L phytagel + 30 g / L sucrose + 1 g / L activated carbon + 8 mg / L 2,4-D + 3 mg / L 2-ip medium for proliferation culture for 30 d. The primary recovery proliferation coefficient can reach 4.5;

[0039] (5) Recovery of callus differentiation ability: After the preservation is completed, transfer the callus to an MS + 30 g / L sucrose + 3.5 g / L phytagel + 1.5 g / L activated carbon + 45 mg / L inositol + 200 mg / L glutamine + 500 mg / L hydrolyzed casein + 0.5 mg / L 2,4-D + 0.3 mg / L 6-BA + 2 mg / L KT + 1 mg / L NAA for callus differentiation culture, and subculture every 30 d. After 3 subcultures, 41% of the callus has the ability to differentiate.

[0040] Example 3

[0041] A method for in vitro conservation of asexual date palm germplasm resources, comprising the following steps

[0042] (1) Preparation of date palm germplasm resources: Transfer the obtained somatic embryos to an MS + 30 g / L sucrose + 3.5 g / L phytagel + 0.5 g / L activated carbon + 0.25 mg / L 2,4-D + 0.1 mg / L NAA medium for date palm somatic embryo germination to obtain test-tube seedlings;

[0043] (2) Preparation of in vitro conservation medium: The conservation medium for test-tube plantlets is 1 / 2MS + 90 g / L sucrose + 30 g / L mannitol + 0.5 mg / L NAA + 3 mg / L chlormequat chloride + 0.5 mg / L paclobutrazol;

[0044] (3) In vitro conservation: Place the callus described in step (1) in the test-tube plantlet conservation medium described in step (2), and culture and conserve it under weak light conditions of 500 Lux at 10°C for 8 months. During this period, the leaves of the test-tube plantlets can remain green and not wither; after the conservation ends, subculture for 20 days can restore growth and induce roots, and the survival rate can reach 80%.

[0045] (4) Rejuvenation and rooting of test-tube plantlets: The test-tube plantlets of date palm can be effectively conserved for 8 months. After the conservation ends, subculture for 20 days can restore growth and induce roots, and the survival rate can reach 80%.

[0046] Example 4

[0047] A method for in vitro conservation of clonal date palm germplasm resources includes the following steps

[0048] (1) Preparation of date palm germplasm resources: Supplement the preparation method of test-tube plantlets; transfer the obtained somatic embryos to MS + 30 g / L sucrose + 3.5 g / L phytagel + 0.5 g / L activated carbon + 0.25 mg / L 2,4-D + 0.1 mg / L NAA for somatic embryo germination of date palm to obtain test-tube plantlets;

[0049] (2) Preparation of in vitro conservation medium: The conservation medium for test-tube plantlets is 1 / 2MS + 90 g / L sucrose + 30 g / L mannitol + 0.5 mg / L NAA + 3 mg / L chlormequat chloride + 0.5 mg / L paclobutrazol;

[0050] (3) In vitro conservation: Place the callus described in step (1) in the test-tube plantlet conservation medium described in step (2), and culture and conserve it under weak light conditions of 1000 Lux at 15°C for 10 months. It can be conserved for 10 months. During this period, the leaves of the test-tube plantlets can remain green and not wither; after the conservation ends, subculture for 30 days can restore growth and induce roots, and the survival rate can reach 80%.

[0051] (4) Rejuvenation and rooting of test-tube plantlets: The test-tube plantlets of date palm can be effectively conserved for 10 months. After the conservation ends, subculture for 30 days can restore growth and induce roots, and the survival rate can reach 80%.

[0052] The various examples in the specification are described in a progressive manner. Each example focuses on the differences from other examples. For the same or similar parts among the various examples, reference can be made to each other.

[0053] The foregoing description of the disclosed embodiments enables those skilled in the art to practice or use the present invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the present invention. Thus, the present invention is not intended to be limited to the embodiments shown herein but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A method for in vitro conservation of date palm germplasm resources of clones, characterized in that, Comprising the following steps (1) Preparation of date palm germplasm resources: After selecting the tissues of date palm for sterilization, they are placed in a culture medium for cultivation to obtain the callus or test-tube plantlets to be preserved; (2) Preparation of in vitro conservation medium: The callus conservation medium is as follows: MS + 30 g / L sucrose + 6 mg / L uniconazole + 1 mg / L 6-BA + 1 mg / L 2,4-D + 3.5 g / L phytagel + 20 g / L mannitol + 0.5 g / L activated carbon; The test-tube plantlet conservation medium is as follows: 1 / 2MS + 90 g / L sucrose + 30 g / L mannitol + 0.5 mg / L NAA + 3 mg / L chlormequat chloride + 0.5 mg / L paclobutrazol; (3) In vitro conservation: The callus described in step (1) is placed in the callus conservation medium described in step (2) for in vitro conservation; Or the test-tube plantlets described in step (1) are placed in the test-tube plantlet conservation medium described in step (2) for in vitro conservation.

2. The in vitro conservation method of a clonal date palm germplasm resource according to claim 1, characterized in that, The preparation process of the callus described in step (1) is as follows: Select the shoot apical meristem of date palm tissue as the explant, and after sterilization, carry out tissue culture to obtain the callus to be preserved.

3. A method for in vitro conservation of a clone date palm germplasm resource according to claim 1, characterized in that The preparation process of the test-tube plantlets described in step (1) is as follows: Select the shoot apical meristem tissue of date palm as the explant, obtain callus through tissue culture, induce the callus into embryogenic callus, the embryogenic callus differentiates into date palm somatic embryos, and the somatic embryos germinate into test-tube plantlets.

4. A method for in vitro conservation of a clonal date palm germplasm resource according to claim 1, characterized in that, The in vitro conservation conditions of the callus described in step (3) are as follows: Dark culture and conservation at 10 - 15 °C.

5. A method for in vitro conservation of a date palm germplasm resource of a clone, characterized in that, The in vitro conservation conditions of the test-tube plantlets described in step (3) are as follows: Culture and conservation under weak light conditions of 500 - 1000 Lux at 10 - 15 °C.

6. The in vitro conservation method of a date palm clone germplasm resource according to claim 1, characterized in that, The conservation time of the callus described in step (3) is 150 d - 180 d; The conservation time of the test-tube plantlets is 8 - 10 months.

7. Application of the in vitro conservation method of clonal date palm germplasm resources according to any one of claims 1 - 6 in the in vitro conservation of clonal date palm germplasm resources.

8. In vitro conservation medium for date palm germplasm resources of clones, characterized in that, Comprising a callus conservation medium and / or a test-tube plantlet conservation medium, the callus conservation medium is as follows: MS + 30 g / L sucrose + 6 mg / L uniconazole + 1 mg / L 6-BA + 1 mg / L 2,4-D + 3.5 g / L phytagel + 20 g / L mannitol + 0.5 g / L activated carbon; The test-tube plantlet conservation medium is as follows: 1 / 2MS + 90 g / L sucrose + 30 g / L mannitol + 0.5 mg / L NAA + 3 mg / L chlormequat chloride + 0.5 mg / L paclobutrazol.

9. Application of the in vitro conservation medium of clonal date palm germplasm resources described in claim 8 in the in vitro conservation of clonal date palm germplasm resources.