In-vitro preservation method for paulownia germplasm resources

Abstract

The invention discloses a paulownia germplasm resource preservation method, which comprises the following steps: A, collecting paulownia seeds, and pretreating the paulownia seeds; b, preparing a culture medium for later use; c, disinfecting the pretreated paulownia seeds, and then putting the paulownia seeds into a culture medium for delayed growth and preservation; d, airing the collected wild paulownia seminal roots, and carrying out root burying breeding in a proper forest land; e, the collected wild paulownia leaves are loaded into a closed collection bag, then the collection bag is filled with silica gel particles, and in-vitro preservation is carried out in a dry environment. The preservation method is easy to operate, the preservation efficiency of the wild germplasm can be effectively improved on the premise that preservation of DNA of the wild paulownia variety is guaranteed, the overall quality of the germplasm is improved, and the survival rate of the wild germplasm is increased. Variety degeneration and disappearance of wild germplasm can be effectively avoided; wherein the adopted culture medium is added with a retardant, meanwhile, the preservation frequency is reduced to the maximum extent, and the production cost is reduced.

Description

Technical Field

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

[0002] Paulownia grows on low-altitude slopes, in forests, valleys and wastelands, either wild or cultivated. Paulownia is distributed in 25 provinces, municipalities and autonomous regions across China. The active ingredients in Paulownia leaves, flowers, bark and roots have antibacterial and antiviral effects; antitussive, expectorant and antiasthmatic effects; effects on the central nervous system; anticancer effects; enhancing insecticide effects; and antihypertensive effects.

[0003] At present, the wild high-quality Paulownia germplasm is gradually disappearing. Because Paulownia seeds are relatively light, it is difficult to have a suitable environment for germination in the wild, and they are prone to mold and deterioration, affecting the germination effect and later growth effect. Therefore, this project collected 400 wild Paulownia germplasms from all over the country. In order to expand the Paulownia germplasm resources, it is necessary to design a method for in vitro preservation of Paulownia germplasm resources. Summary of the invention

[0004] The object of the present invention is to provide a method for in vitro preservation of Paulownia germplasm resources to solve the problems raised in the above background technology.

[0005] To achieve the above object, the present invention provides the following technical solution: a method for in vitro preservation of Paulownia germplasm resources, the preservation method comprising the following steps:

[0006] A. Collecting Paulownia seeds and pre-treating the Paulownia seeds;

[0007] B. Prepare culture medium for standby use;

[0008] C. Sterilize the pretreated Paulownia seeds and then place them in a culture medium to delay their growth and preserve them;

[0009] D. Dry the collected wild Paulownia seed roots and bury them in suitable forest land for seed breeding;

[0010] E. Put the collected wild Paulownia leaves into a sealed collection bag, fill it with silica gel particles, and store them in vitro in a dry environment.

[0011] Preferably, the culture medium components in step B include 30-50 parts of brewer's yeast dry residue, 10-20 parts of yeast extract, 2-4 parts of peptone, 4-10 parts of hot-pressed bean powder, 3-9 parts of mung bean sprout grinding liquid and 30-40 parts of deionized water, and 3-6 parts of retardant chlormequat chloride CCC in parts by weight.

[0012] Preferably, the specific culture method of the culture medium in step C is as follows:

[0013] a. Place the sterilized Paulownia seeds on the culture medium;

[0014] b. Control the culture temperature to 25°C-28°C, with 16 hours of light and 8 hours of darkness during the culture process; c. After the seeds germinate, the culture of Paulownia seed germination is completed.

[0015] Preferably, after the seeds germinate in step c, they are subcultured and preserved in tissue culture bottles.

[0016] Compared with the prior art, the beneficial effects of the present invention are as follows: the preservation method adopted by the present invention is simple and convenient to operate, and can effectively improve the preservation efficiency of wild germplasm while ensuring the preservation of wild Paulownia varieties' DNA, thereby improving the overall quality of the germplasm and effectively avoiding the variety degeneration and disappearance of wild germplasm; wherein, the culture medium adopted is added with a retardant, while minimizing the number of preservation times and reducing production costs. DETAILED DESCRIPTION

[0017] The technical solutions in the embodiments of the present invention are described clearly and completely below. 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.

[0018] Embodiment 1:

[0019] The present invention provides the following technical solution: a method for in vitro preservation of Paulownia germplasm resources, the preservation method comprising the following steps:

[0020] A. Collecting Paulownia seeds and pre-treating the Paulownia seeds;

[0021] B. Prepare culture medium for standby use;

[0022] C. Sterilize the pretreated Paulownia seeds and then place them in a culture medium to delay their growth and preserve them;

[0023] D. Dry the collected wild Paulownia seed roots and bury them in suitable forest land for seed breeding;

[0024] E. Put the collected wild Paulownia leaves into a sealed collection bag, fill it with silica gel particles, and store them in vitro in a dry environment.

[0025] In this embodiment, the culture medium components in step B include 30 parts of brewer's yeast dry residue, 10 parts of yeast extract, 2 parts of peptone, 4 parts of hot-pressed bean powder, 3 parts of mung bean sprout grinding liquid, 30 parts of deionized water, and 3 parts of retardant chlormequat CCC by weight.

[0026] In this embodiment, the specific culture method of the culture medium in step C is as follows:

[0027] a. Place the sterilized Paulownia seeds on the culture medium;

[0028] b. Control the culture temperature to 25°C, with 16 hours of light and 8 hours of darkness during the culture process;

[0029] c. After the seeds germinate, the cultivation of Paulownia seed germination is completed.

[0030] In this embodiment, after the seeds germinate in step c, they are subcultured and preserved in tissue culture bottles.

[0031] Embodiment 2:

[0032] A method for in vitro preservation of Paulownia germplasm resources, the preservation method comprising the following steps:

[0033] A. Collecting Paulownia seeds and pre-treating the Paulownia seeds;

[0034] B. Prepare culture medium for standby use;

[0035] C. Sterilize the pretreated Paulownia seeds and then place them in a culture medium to delay their growth and preserve them;

[0036] D. Dry the collected wild Paulownia seed roots and bury them in suitable forest land for seed breeding;

[0037] E. Put the collected wild Paulownia leaves into a sealed collection bag, fill it with silica gel particles, and store them in vitro in a dry environment.

[0038] In this embodiment, the components of the culture medium in step B include 50 parts of brewer's yeast dry residue, 20 parts of yeast extract, 4 parts of peptone, 10 parts of hot-pressed bean powder, 9 parts of mung bean sprout grinding liquid, 40 parts of deionized water, and 6 parts of the retarder chlormequat CCC by weight.

[0039] In this embodiment, the specific culture method of the culture medium in step C is as follows:

[0040] a. Place the sterilized Paulownia seeds on the culture medium;

[0041] b. Control the culture temperature to 28°C, with 16 hours of light and 8 hours of darkness during the culture process;

[0042] c. After the seeds germinate, the cultivation of Paulownia seed germination is completed.

[0043] In this embodiment, after the seeds germinate in step c, they are subcultured and preserved in tissue culture bottles.

[0044] Embodiment three:

[0045] A method for in vitro preservation of Paulownia germplasm resources, the preservation method comprising the following steps:

[0046] A. Collecting Paulownia seeds and pre-treating the Paulownia seeds;

[0047] B. Prepare culture medium for standby use;

[0048] C. Sterilize the pretreated Paulownia seeds and then place them in a culture medium to delay their growth and preserve them;

[0049] D. Dry the collected wild Paulownia seed roots and bury them in suitable forest land for seed breeding;

[0050] E. Put the collected wild Paulownia leaves into a sealed collection bag, fill it with silica gel particles, and store them in vitro in a dry environment.

[0051] In this embodiment, the components of the culture medium in step B include 35 parts of brewer's yeast dry residue, 12 parts of yeast extract, 3 parts of peptone, 5 parts of hot-pressed bean powder, 4 parts of mung bean sprout grinding liquid, 32 parts of deionized water, and 4 parts of retardant chlormequat CCC.

[0052] In this embodiment, the specific culture method of the culture medium in step C is as follows:

[0053] a. Place the sterilized Paulownia seeds on the culture medium;

[0054] b. Control the culture temperature to 26°C, with 16 hours of light and 8 hours of darkness during the culture process;

[0055] c. After the seeds germinate, the cultivation of Paulownia seed germination is completed.

[0056] In this embodiment, after the seeds germinate in step c, they are subcultured and preserved in tissue culture bottles.

[0057] Embodiment 4:

[0058] A method for in vitro preservation of Paulownia germplasm resources, the preservation method comprising the following steps:

[0059] A. Collecting Paulownia seeds and pre-treating the Paulownia seeds;

[0060] B. Prepare culture medium for standby use;

[0061] C. Sterilize the pretreated Paulownia seeds and then place them in a culture medium to delay their growth and preserve them;

[0062] D. Dry the collected wild Paulownia seed roots and bury them in suitable forest land for seed breeding;

[0063] E. Put the collected wild Paulownia leaves into a sealed collection bag, fill it with silica gel particles, and store them in vitro in a dry environment.

[0064] In this embodiment, the culture medium components in step B include 45 parts of brewer's yeast dry residue, 18 parts of yeast extract, 3 parts of peptone, 8 parts of hot-pressed bean powder, 8 parts of mung bean sprout grinding liquid, 38 parts of deionized water, and 5 parts of retardant chlormequat CCC by weight.

[0065] In this embodiment, the specific culture method of the culture medium in step C is as follows:

[0066] a. Place the sterilized Paulownia seeds on the culture medium;

[0067] b. Control the culture temperature to 27°C, with 16 hours of light and 8 hours of darkness during the culture process;

[0068] c. After the seeds germinate, the cultivation of Paulownia seed germination is completed.

[0069] In this embodiment, after the seeds germinate in step c, they are subcultured and preserved in tissue culture bottles.

[0070] Embodiment five:

[0071] A method for in vitro preservation of Paulownia germplasm resources, the preservation method comprising the following steps:

[0072] A. Collecting Paulownia seeds and pre-treating the Paulownia seeds;

[0073] B. Prepare culture medium for standby use;

[0074] C. Sterilize the pretreated Paulownia seeds and then place them in a culture medium to delay their growth and preserve them;

[0075] D. Dry the collected wild Paulownia seed roots and bury them in suitable forest land for seed breeding;

[0076] E. Put the collected wild Paulownia leaves into a sealed collection bag, fill it with silica gel particles, and store them in vitro in a dry environment.

[0077] In this embodiment, the culture medium components in step B include 40 parts of brewer's yeast dry residue, 15 parts of yeast extract, 3 parts of peptone, 7 parts of hot-pressed bean powder, 6 parts of mung bean sprout grinding liquid, 35 parts of deionized water, and 5 parts of retardant chlormequat CCC by weight.

[0078] In this embodiment, the specific culture method of the culture medium in step C is as follows:

[0079] a. Place the sterilized Paulownia seeds on the culture medium;

[0080] b. Control the culture temperature to 27°C, with 16 hours of light and 8 hours of darkness during the culture process;

[0081] c. After the seeds germinate, the cultivation of Paulownia seed germination is completed.

[0082] In this embodiment, after the seeds germinate in step c, they are subcultured and preserved in tissue culture bottles.

[0083] Experimental example:

[0084] The germination experiment was conducted using the Paulownia seeds preserved in the embodiments of the present invention and compared with the conventionally preserved seeds. The data obtained are shown in the following table:

[0085] Germination rate (%) Regular save 45.5 Embodiment 1 79.2 Embodiment 2 80.5 Embodiment 3 80.9 Embodiment 4 81.2 Embodiment 5 82.8

[0086] In summary, the preservation method adopted in the present invention is simple and convenient to operate. It can effectively improve the preservation efficiency of wild germplasm and improve the overall quality of germplasm while ensuring the preservation of wild Paulownia varieties' DNA, and can effectively avoid the variety degeneration and disappearance of wild germplasm. Among them, the culture medium used is added with a retardant, while minimizing the number of preservation times and reducing production costs.

[0087] Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions and variations may be made to the embodiments without departing from the principles and spirit of the present invention, and that the scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. A method for preserving Paulownia germplasm resources, characterized in that: The saving method includes the following steps: A. Collecting Paulownia seeds and pre-treating the Paulownia seeds; B. Prepare culture medium for standby use; C. Sterilize the pretreated Paulownia seeds and then place them in a culture medium to delay their growth and preserve them; D. Dry the collected wild Paulownia seed roots and bury them in suitable forest land for seed breeding; E. Put the collected wild Paulownia leaves into a sealed collection bag, fill it with silica gel particles, and store them in vitro in a dry environment.

2. The in vitro preservation method of Paulownia germplasm resources according to claim 1, characterized in that: The culture medium components in step B include, by weight, 30-50 parts of brewer's yeast dry residue, 10-20 parts of yeast extract, 2-4 parts of peptone, 4-10 parts of hot-pressed bean powder, 3-9 parts of mung bean sprout grinding liquid, 30-40 parts of deionized water, and 3-6 parts of retardant chlormequat chloride CCC.

3. The in vitro preservation method of Paulownia germplasm resources according to claim 1, characterized in that: The specific culture method of the culture medium in step C is as follows: a. Place the sterilized Paulownia seeds on the culture medium; b. Control the culture temperature to 25℃-28℃, with 16 hours of light and 8 hours of darkness during the culture process; c. After the seeds germinate, the cultivation of Paulownia seed germination is completed.

4. The in vitro preservation method of Paulownia germplasm resources according to claim 3, characterized in that: After the seeds germinate in step c, they are subcultured and preserved in tissue culture bottles.