阅读说明：本技术 一种裙带菜成熟孢子囊叶的保存方法 (Method for preserving mature sporangium leaves of undaria pinnatifida ) 是由 李静 逄少军 苏丽 李晓东 单体锋 于 2021-06-16 设计创作，主要内容包括：本发明涉及经济褐藻裙带菜种质资源收集及育种领域,具体的说是一种裙带菜成熟孢子囊叶的保存方法。将裙带菜成熟的孢子囊叶用滤纸擦拭,去除孢子囊叶表面多余的水分,再用干净的滤纸包裹,放入到样品袋中密封,随后放置到4℃条件下保存数十天后,仍能采集得到裙带菜正常的游孢子。该保存方法的建立有利于进一步保证裙带菜种质资源的收集、保存及种苗培育工作的顺利进行。(The invention relates to the field of collection and breeding of economic brown algae undaria pinnatifida germplasm resources, in particular to a method for preserving mature sporangium leaves of undaria pinnatifida. Wiping mature sporangium leaves of the undaria pinnatifida with filter paper, removing excessive water on the surfaces of the sporangium leaves, wrapping with clean filter paper, sealing in a sample bag, and then storing at 4 ℃ for tens of days to obtain normal zoospores of the undaria pinnatifida. The establishment of the preservation method is beneficial to further ensuring the smooth operation of collection and preservation of the germplasm resources of the undaria pinnatifida and the cultivation of seedlings.)

1. A preservation method of mature sporocyst leaf of undaria pinnatifida is characterized in that: wiping mature sporangium leaves of the undaria pinnatifida with filter paper to enable the surfaces of the mature sporangium leaves to reach a state without excessive moisture, wrapping the wiped sporangium leaves with the filter paper, putting the wrapped sporangium leaves into a sample bag for sealing, then storing the sample bag at a low temperature for 5 to 20 days, and taking out the sample bag to collect normal free spores.

2. The method for preserving mature sporangium leaf of undaria pinnatifida according to claim 1, wherein:

when the filter paper is used for wrapping, the sporangium leaves of each individual undaria pinnatifida are wrapped together.

3. The method for preserving mature sporangium leaf of undaria pinnatifida according to claim 1, wherein:

the mature sporangium leaf refers to the sporocyst leaf of the undaria pinnatifida which forms mature sporangium and can successfully release the zoospores.

4. The method for preserving mature sporangium leaf of undaria pinnatifida according to claim 1, wherein: the low-temperature preservation is preservation at 4 ℃.

5. The method for preserving mature sporocyst leaf of undaria pinnatifida according to any one of claims 1 to 4, wherein: taking out after low-temperature preservation, washing the surface of the sporocyst by sterile seawater to remove the sporocyst group with damaged surface layer, immersing the sporocyst leaf into the sterile seawater after washing, and collecting the zoospores after 0.5-2 h.

6. The method for preserving mature sporangium leaf of undaria pinnatifida according to claim 5, wherein: collecting zoospore, placing in 1/2 PES-containing culture medium, and culturing at 8-12 deg.C under light intensity of 5-15 μmol phosns m^-2s^-1Culturing under the condition that the photoperiod is 12:12(L: D), can realize vegetative growth and long-term storage; placing in PES-containing medium at 15-18 deg.C under light intensity of 30-40 μmol photons m^-2s^-1Culturing under the condition of photoperiod of 12:12(L: D) until the female and male gametophytes form oocysts and seminal vesicles, and completing the fertilization process to form juvenile sporophytes.

Technical Field

The invention relates to the field of collection and breeding of economic brown algae undaria pinnatifida germplasm resources, in particular to a method for preserving mature sporangium leaves of undaria pinnatifida.

Background

Undaria pinnatifida is important economic seaweed in China, the annual output is 17 ten thousand tons (dry weight), and the undaria pinnatifida has wide application in the fields of chemical industry, medicine, aquatic product bait and the like except the edible value. Undaria pinnatifida is annual seaweed, and the life history of the undaria pinnatifida is divided into a large sporophyte generation and a microscopic gametophyte generation. The large sporophyte develops in the stem part to form sporangium leaf during sexual maturity, and the sporangium leaf is the reproductive organ of undaria pinnatifida and can form a large amount of sporangium. The sporangium can generate free spores through meiosis, and the free spores are attached to germinate into female and male gametophytes after being diffused. The microscopic gametophyte is a main form for preserving the germplasm of the undaria pinnatifida and is also an important resource for developing the crossbreeding work of the undaria pinnatifida. Therefore, the successful collection of the zoospores is the first task to continuously collect and store the germplasm resources of the undaria pinnatifida. In actual work, the undaria pinnatifida needs to go to a natural distribution and cultivation sea area for collecting mature sporangium leaves, and then reaches a conditioned room through a transportation mode for free spore release and collection. Sometimes, due to weather or traffic reasons, the sporangium leaves cannot arrive indoors in time to carry out collection work, so that the sporangium leaves are rotted and deteriorated, collection and further preservation of germplasm resources cannot be completed smoothly, and waste of manpower and financial resources is caused. In order to avoid the above situations, it is urgently needed to establish an effective method for preserving the sporocyst leaf of undaria pinnatifida so as to ensure the success of the collection work of the germplasm resources of undaria pinnatifida, and further have important significance for the preservation of the germplasm resources and the development of the seedling cultivation work.

Disclosure of Invention

The invention aims to provide a method for preserving mature sporangium leaves of undaria pinnatifida.

In order to achieve the purpose, the invention adopts the technical scheme that:

a method for preserving mature sporangium leaf of Undaria pinnatifida comprises wiping mature sporangium leaf of Undaria pinnatifida with filter paper to make its surface reach a state without excessive water, wrapping the wiped sporangium leaf with filter paper, sealing in a sample bag, preserving at low temperature for 5-20 days, and taking out to obtain normal free spores.

Wherein, the method of storing the sporocyst leaf of the undaria pinnatifida in a sealing way at low temperature can effectively delay the rotting and dehydration of the sporocyst leaf and achieve the purpose of prolonging the storage life.

When the filter paper is used for wrapping, the sporangium leaves of each individual undaria pinnatifida are wrapped together.

The mature sporangium leaf refers to the sporocyst leaf of the undaria pinnatifida which forms mature sporangium and can successfully release the zoospores.

The low-temperature preservation is preservation at 4 ℃.

Taking out after low-temperature preservation, washing the surface of the sporocyst by sterile seawater to remove the sporocyst group with damaged surface layer, immersing the sporocyst leaf into the sterile seawater after washing, and collecting the zoospores after 0.5-2 h.

Collecting zoospore, placing in 1/2 PES-containing culture medium, and culturing at 8-12 deg.C under light intensity of 5-15 μmol phosns m^{- 2}s^-1Culturing under the condition that the photoperiod is 12:12(L: D), can realize vegetative growth and long-term storage; placing in PES-containing medium at 15-18 deg.C under light intensity of 30-40 μmol photons m^-2s^-1Culturing under the condition of photoperiod of 12:12(L: D) until the female and male gametophytes form oocysts and seminal vesicles, and completing the fertilization process to form juvenile sporophytes.

The zoospore is the germ cell of the undaria pinnatifida. Gametophytes can be formed after the natatoria germinates, and can grow nutritionally under the condition of low temperature and low light and be stored for a long time; by increasing the temperature and light intensity, the gametophyte development can be promoted, and the fertilization process is completed to form juvenile sporophytes.

Furthermore, the gametophyte grows nutritionally, so that the long-term storage of the undaria pinnatifida germplasm resources can be realized; the gametophyte is developed and fertilized to form juvenile sporophyte, so that the cultivation process of the undaria pinnatifida seedlings can be completed.

The thallus laminariae gametophyte is subjected to vegetative growth in 1/2PES culture medium at 8-12 deg.C under light intensity of 5-15 μmol photons m^-2s^-1Culturing under the condition that the photoperiod is 12:12(L: D); the gametophyte of Undaria Pinnatifida is required to be placed in PES culture medium at 15-18 deg.C under light intensity of 30-40 μmol photons m^-2s^-1Culturing under the condition of photoperiod of 12:12(L: D) until the female and male gametophytes form oocysts and seminal vesicles, and completing the fertilization process to form juvenile sporophytes.

The sterilized seawater is natural seawater sterilized at 121 deg.C and 0.1 Mpa.

The invention has the advantages that:

the method is implemented by taking the marine alga undaria pinnatifida as an implementation object, is a transient preservation method of the reproductive organs of living algae, takes the special living environment and the propagation characteristics of the marine algae into full consideration in the implementation and application stage, and has the characteristics of simple and convenient operation and strong practicability.

The survival medium of the undaria pinnatifida is seawater, the natural population of the undaria pinnatifida is distributed in the sea intertidal zone or the sub-tidal zone, the culture population is distributed in the coastal culture area, and compared with other species (such as land plants, animals and the like), the undaria pinnatifida has obvious difference and great difficulty in the aspects of collection and preservation of germplasm resources. If the undaria pinnatifida forms mature reproductive organs (sporangium leaves), and the sporangium leaves are dehydrated for a long time without treatment, the phenomena of dehydration, decay and death are easy to occur; the re-hydration of sporangium leaves after leaving water will result in the concentrated and great diffusion of germ cells (free spores). Therefore, once the mature sporangia of the undaria pinnatifida leaves water, the mature sporangia cannot be put into seawater again for culture before collecting free spores, and if the sporangia leaves leave water for a long time, specific conditions are required to be set so as to effectively prolong the life of the sporangia.

Further, the present invention fully considers the occurrence of rotting phenomenon during the preservation of sporangium leaves due to the above-mentioned problems. The cleaned sporangium leaves are put at the low temperature of 4 ℃ to effectively delay the occurrence of the rotten phenomenon of the sporangium leaves.

The invention fully considers the dehydration phenomenon in the preservation process of the sporangium leaves. The filter paper is adopted for wrapping, and the spore capsule leaves are placed in the sample bag for sealed storage, so that dehydration of the spore capsule leaves is effectively avoided.

Drawings

FIG. 1 is a diagram showing the states of sporangium leaves after 3 days of storage under different storage conditions according to an embodiment of the present invention.

FIG. 2 shows the germination rate of the zoospores released from sporocysts and the fertilization rate of the gametophytes developed by the sporocysts stored for different days under the storage condition 1 provided in the example of the present invention.

Detailed Description

The following examples are presented to further illustrate embodiments of the present invention, and it should be understood that the embodiments described herein are for purposes of illustration and explanation only and are not intended to limit the invention.

Example 1

Mature undaria pinnatifida sporocyst leaves collected in the Dalian sea area are wiped on the surface of the mature undaria pinnatifida sporocyst leaves by using filter paper, residual moisture and attachments (including silt, attached foreign algae and the like) on the surface are removed to the extent that the surface has no excessive moisture, and then the mature undaria pinnatifida sporocyst leaves are respectively subjected to storage treatment under different conditions and days (see table 1).

The different conditions are as follows:

condition 1: wiping the sporangium leaves with filter paper to the extent that the surface has no redundant water, wrapping the sporangium leaves with the filter paper, putting the wrapped sporangium leaves into a sample bag, sealing the sample bag, and storing the sample bag at 4 ℃ for different days;

condition 2: wiping the sporangium leaves with filter paper to the extent that no redundant water exists on the surface, wrapping the sporangium leaves with the filter paper, putting the wrapped sporangium leaves into a sample bag, sealing the sample bag, and storing the sample bag for 3 days at 18 ℃;

condition 3: the sporangial leaves were wiped with filter paper until the surface had no excess water, and then directly stored at 4 ℃ for 3 days.

Table 1 shows the results of the storage treatment under different conditions and days

Storage conditions	Condition setting	Days of storage
			1	4 ℃ sealing	3、10、18
2	18 ℃ sealing	3
			3	4 ℃ without sealing	3

Taking out preserved Undaria pinnatifida sporocyst leaf, washing surface with sterilized seawater, removing surface damaged sporocyst group, placing into sterilized seawater, diffusing for 0.5-2 hr, sucking free spore liquid into culture dish containing PES culture solution and having diameter of 9cm, collecting 3 culture dishes, placing at 15-18 deg.C, and placing light intensity of 30-40 μmol photons m^-2s^-1After culturing for 24 hours under the condition that the photoperiod is 12:12(L: D), counting the germination rate of the zoospores; and after the cultivation is continued for 14-20 days, counting the fertilization rate of the gametophyte. Meanwhile, the zoospores released from the sporocyst leaves (preserved for 0 day) which are not preserved are collected, and the germination rate and the fertilization rate of the gametophyte cultured in the later period are used as experimental controls.

The statistical method of the germination rate comprises the following steps: the petri dish was placed under an inverted microscope at 40 × 10 magnification, and the percentage of germinated free spores to the total number of free spores in the field was counted, for a total of 5 fields. The germinated zoospores are those that appear in the germination tubes.

The statistical method of the fertilization rate comprises the following steps: the dishes were placed under an inverted microscope and at a magnification of 10X 10, the percentage of fertilized eggs in the field of view to the total number of discharged eggs was counted, for a total of 5 fields (see FIGS. 1 and 2).

The results are as follows:

the sporangial leaves exhibited distinctly different states when stored under different storage conditions for 3 days (as shown in FIG. 1).

Storage condition 1 (fig. 1, a): the sporangium has good state, no obvious water loss and rot phenomenon, the water loss rate is 7.072.00 percent, and the free spores can be successfully released.

Storage condition 2 (fig. 1, b): the sporangium is obviously rotten, the water loss rate is 11.05 +/-1.90 percent, and the free spores cannot be released.

Storage condition 3 (fig. 1, c): the sporangium leaves are seriously dehydrated, the water loss rate is as high as 70.04 +/-1.43 percent, and the zoospores cannot be released.

The sporocyst leaves stored for 3, 10 and 18 days under the storage condition 1 successfully released the zoospores, and the results of the germination rate of the zoospores and the fertilization rate of the gametophytes formed by development are shown in fig. 2.

The results show that the germination rate of collected nataspore gradually decreases with increasing number of cold days of sporangium leaves after storage for more than a certain number of cold days (3 days). There was no significant difference in germination rate between the free spores collected on sporocyst leaves that were refrigerated for 3 days and those collected on sporocyst leaves that were not refrigerated, while the germination rate of the free spores collected on sporocyst leaves that were refrigerated for 10 and 18 days was significantly reduced. However, the germinated free spores can basically complete the subsequent development and fertilization processes, so that no significant difference exists among cold storage days in terms of fertilization rate.

The sporozoites are in a survival state if the sporozoites can germinate smoothly, and gametophytes formed by the germinated sporozoites can continuously grow nutritionally by providing proper culture conditions, so that the aim of long-term preservation of the undaria pinnatifida germplasm resources is fulfilled; and the gametophyte formed by the germination of the zoospore can complete the development and fertilization processes by providing development conditions, so that the juvenile sporophyte is formed, and the aim of seedling cultivation is fulfilled. The method has important practical significance for collection and preservation of undaria pinnatifida germplasm resources and seedling cultivation. The preservation method can be used in marine collection of sporocyst leaf of Undaria pinnatifida, and is also suitable for other economic seaweed species of sexual maturity Laminariales, such as Laminaria japonica.