CN104126504A - Culture method for aseptic seedling of Zostera marina - Google Patents
Culture method for aseptic seedling of Zostera marina Download PDFInfo
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- CN104126504A CN104126504A CN201310431215.4A CN201310431215A CN104126504A CN 104126504 A CN104126504 A CN 104126504A CN 201310431215 A CN201310431215 A CN 201310431215A CN 104126504 A CN104126504 A CN 104126504A
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Abstract
The invention relates to a culture method for an aseptic seedling of Zostera marina. The culture method comprises the following steps: selecting plump and intact seeds of Zostera marina, placing the seeds in a sterilization container in an aseptic environment and washing the seeds with sterilized natural seawater 3 to 5 times; soaking the seeds with 75% ethanol for 30 min and then washing the seeds with sterilized natural seawater 3 to 5 times; transferring the seeds to a hydrogen dioxide solution with a concentration of 10% for soaking for 20 min and then washing the seeds with sterilized natural seawater 3 to 5 times; dispersedly inoculating the seeds on a solid medium and carrying out culture in a constant-temperature illumination incubator where temperature is 18 DEG C, light intensity is 1500 lux and a ratio of illumination to darkness is 12 h: 12 h; carrying out observation day by day and transferring and inoculating uncontaminated seeds in time in case of contamination; and when the length of the seedlings is 1 to 2 cm, transferring the seedlings into a liquid medium for culture or taking related tissue from the seedlings for culture. According to the invention, solid culture is carried out on the seeds of Zostera marina, uncontaminated seeds can be transferred and inoculated to a novel medium in time, so the phenomenon that one contaminated seed leads to loss of the whole bottle of seeds is avoided, and a contamination rate is effectively reduced.
Description
Technical field
Sea grass tissue culture technique field under the present invention, relates to a kind of cultural method of zostera aseptic seedling.
Background technology
Zostera marina (
zostera marinal.) be marine site, temperate zone, Northern Hemisphere distribution sea grass the most widely, there is important Ecosystem Service.Due to its population large area degeneration in the world in recent decades, so its population reconstruction work has become many coastal states' important ecological engineering project, in reconstruction, need a large amount of provenances, the collection of provenance often can cause again the further destruction in its former habitat.Therefore,, if utilize tissue culture technique to turn out fast, in large quantity cheap pure Zostera marina test-tube plantlet indoor, can meet the wilderness demand to provenance in its population reconstruction.
But research shows, due to Zostera marina leaf epidermal cell anoderm, and be scattered with a large amount of air flues in leaf and stem tissue, in root, also there are many gas cavity, so while making the explant taking wild adult plant as tissue cultivation, decontaminant concentration and disinfecting time be difficult grasp extremely, and normal appearance pollutes or sterilized and kill the dilemma of most tissues not because sterilizing.And the seed coat of grass wrack seeds is hard, abundant, the tolerance of disinfectant is obviously better than to the tissues such as root, stem, leaf; And seedling physiological age is the youngest, so the best explant that aseptic seedling is Zostera marina tissue to be cultivated is selected.
In July, 2010 Chinese Marine University open " a kind of breeding method of zostera aseptic seedlings ", open " a kind of method of the cultivating zostera aseptic seedlings " patent of in January, 2012 Shandong Oriental Ocean Sci-tech Co., Ltd., two patents are the cultural method of zostera aseptic seedling, although seed disinfection mode is different with culture medium, also successfully obtained zostera aseptic seedling, but the two is all cultivated grass wrack seeds with liquid medium, a large amount of tissues is cultivated practice and is shown, multiple explants are placed in same liquid nutrient medium and cultivate, very easily cause pollution, although grass wrack seeds is with respect to its root, stem, leaf more easily reaches thorough disinfection, but its seed is less, on seed coat, there is 10-20 bar to indulge rib, so have unavoidably the halfway seed of sterilization after sterilization, if they are carried out to liquid culture, often there will be because of a serious consequence that seed contamination causes one bottle of seed all to pollute in blake bottle.
Summary of the invention
The object of the invention is: the cultural method that a kind of zostera aseptic seedling of effective minimizing pollution rate is provided.Its feature comprises the following steps:
(1) choose full complete, the grass wrack seeds that seed coat presents brown of form, under the gnotobasis of superclean bench, be placed in sterilized glass container, rinse 3-5 time with sterilizing natural sea-water, water is blotted with sterilizing filter paper;
(2) being 75% ethanolic solution by concentration soaks seed after 30 seconds, then with sterilizing natural sea-water flushing 3-5 time, water is blotted with sterilizing filter paper;
(3) grass wrack seeds being moved to concentration is to soak 20 minutes in 10% hydrogenperoxide steam generator, then rinses 3-5 time with sterilizing natural sea-water;
(4) seed of sterilization is disperseed to be inoculated on solid culture medium;
(5) put into constant temperature illumination box and cultivate, condition of culture is: temperature keeps 18 DEG C, intensity of illumination 1500lux, illumination: dark=12h:12h;
(6) observe by sky, find that there is polluter and immediately uncontaminated person is transferred in new medium;
(7), when seedling grows to 1 ~ 2cm length, can be proceeded in corresponding liquid nutrient medium and cultivate, or directly Qu Qi linked groups organizes cultivation.
The invention has the advantages that: adopt solid culture medium to cultivate Zostera marina disinfection seed, if any indivedual seed contaminations, due to the stationarity of solid culture medium, uncontaminated seed can be transferred on new medium in time, avoid a phenomenon appearance (Fig. 1) of polluting and lose whole bottle seed.Therefore the present invention greatly reduces the pollution rate in Zostera marina Aseptic seedling procurement process, saves seed, cost-saving, improves the productive rate of aseptic seedling.
Brief description of the drawings
When Fig. 1 adopts liquid culture, Zostera marina seedling occurs that whole bottle pollutes photo
The Zostera marina seedling photo that Fig. 2 has just germinateed on solid culture medium
Fig. 3 proceeds to the seedling photo in liquid nutrient medium
Embodiment
The cultural method of the zostera aseptic seedling of a kind of effective minimizing pollution rate of the present invention comprises following examples, and the following examples can further illustrate the present invention, but do not limit the present invention in any way.
embodiment 1:
a cultural method for zostera aseptic seedling, implementation step comprises:
(1) choose full complete, the seed coat of the form of just having adopted back August from marine site and be pitchy, great-hearted grass wrack seeds;
(2) first with natural sea-water, the attachment of the surface of the seed and impurity are rinsed well, under the gnotobasis of superclean bench, be placed in sterilized glass container, rinse 3-5 time with sterilizing natural sea-water, water is blotted with sterilizing filter paper;
(3) being 75% ethanolic solution by concentration soaks seed after 30 seconds, then with sterilizing natural sea-water flushing 3-5 time, water is blotted with sterilizing filter paper;
(4) grass wrack seeds being moved to concentration is immersion 20 minutes in 10% hydrogenperoxide steam generator (containing 0.1% tween, hydrogenperoxide steam generator dilutes preparation with sterilizing seawater), then with sterilizing natural sea-water flushing 3-5 time, water is blotted with sterilizing filter paper;
(5) seed of sterilizing is disperseed to be inoculated in the culture dish that solid culture medium is housed, medium is only containing 4g/L agar and 10g/L sea salt, and culture dish seals with parafilm sealed membrane;
(6)put into constant temperature illumination box and cultivate, condition of culture is: temperature keeps 18 DEG C, intensity of illumination 1500lux, illumination: dark=12h: 12h.
(7) observe by sky, find that there is polluter and immediately uncontaminated person is transferred in new culture dish;
After (8) 20 days, seed germinates successively, as shown in Figure 2, after 30 days, 80% seed sprouting, germinateed after 1 week, it is long that seedling can grow to 1-2cm, now can be proceeded to and in corresponding liquid nutrient medium, cultivate (Fig. 3), or directly Qu Qi linked groups organizes cultivation.
The present embodiment sea salt used is that the large seawater in general sea, Qingdao have the seawater extract that limit company produces; Constant temperature illumination box is Shanghai rich news SPX-250B-G type illumination box.
embodiment 2:
(1) choose full complete, the seed coat of the form of just having adopted back July from marine site and be pitchy, great-hearted grass wrack seeds;
(2) first with natural sea-water, attachment and impurity are rinsed well, under the gnotobasis of superclean bench, be placed in sterilized glass container, rinse 3-5 time with sterilizing natural sea-water, water is blotted with sterilizing filter paper;
(3) being 75% ethanolic solution by concentration soaks seed after 30 seconds, then with sterilizing natural sea-water flushing 3-5 time, water is blotted with sterilizing filter paper;
(4) grass wrack seeds being moved to concentration is immersion 20 minutes in 10% hydrogenperoxide steam generator (containing 0.1% tween, hydrogenperoxide steam generator dilutes preparation with sterilizing seawater), then with sterilizing natural sea-water flushing 3-5 time, water is blotted with sterilizing filter paper;
(5) seed of sterilizing is disperseed to be inoculated in the culture dish that solid culture medium is housed, medium is the 1/2MS medium containing 4g/L agar and 10g/L sea salt, parafilm sealed membrane sealing for culture dish;
(6)put into constant temperature illumination box and cultivate, condition of culture is: temperature keeps 18 DEG C, intensity of illumination 1500lux, illumination: dark=12h: 12h.
(7) observe by sky, find that there is polluter and immediately uncontaminated person is transferred in new culture dish;
After (8) 20 days, seed germinates successively, as shown in Figure 1, after 30 days, 80% seed sprouting, germinateed after 1 week, it is long that seedling can grow to 1-2cm, now can be proceeded in corresponding liquid nutrient medium and cultivate, or directly Qu Qi linked groups organizes cultivation.
The present embodiment sea salt used is that the large seawater in general sea, Qingdao have the seawater extract that limit company produces; Constant temperature illumination box is Shanghai rich news SPX-250B-G type illumination box.
embodiment 3:
(1) choose full complete, the seed coat of the form of just having adopted back July from marine site and be pitchy, great-hearted grass wrack seeds, be placed in the blake bottle of 250ml, add natural sea-water 200ml, be put at 4 DEG C of Refrigerator stores, without inflation and illumination, change seawater every day twice, the seawater of replacing needs precooling.
(2) preservation 15 days or the seed of longer time are first rinsed well attachment and impurity with natural sea-water, under the gnotobasis of superclean bench, be placed in sterilized glass container, rinse 3-5 time with sterilizing natural sea-water, water is blotted with sterilizing filter paper;
(3) being 75% ethanolic solution by concentration soaks seed after 30 seconds, then with sterilizing natural sea-water flushing 3-5 time, water is blotted with sterilizing filter paper;
(4) grass wrack seeds being moved to concentration is immersion 20 minutes in 10% hydrogenperoxide steam generator (containing 0.1% tween, hydrogenperoxide steam generator dilutes preparation with sterilizing seawater), then with sterilizing natural sea-water flushing 3-5 time, water is blotted with sterilizing filter paper;
(5) seed of sterilizing is disperseed to be inoculated in the culture dish that solid culture medium is housed, medium is only containing 4g/L agar and 10g/L sea salt, and culture dish seals with parafilm sealed membrane;
(6)put into constant temperature illumination box and cultivate, condition of culture is: temperature keeps 18 DEG C, intensity of illumination 1500lux, illumination: dark=12h: 12h.
(7) observe by sky, find that there is polluter and immediately uncontaminated person is transferred in new culture dish;
After (8) 5 days, seed germinates successively, and after 10 days, 90% seed sprouting, germinateed after 1 week, and it is long that seedling can grow to 1-2cm, now can be proceeded in corresponding liquid nutrient medium and cultivate, or directly Qu Qi linked groups organizes cultivation.
The present embodiment sea salt used is that the large seawater in general sea, Qingdao have the seawater extract that limit company produces; Constant temperature illumination box is Shanghai rich news SPX-250B-G type illumination box.
Claims (1)
1. a cultural method for zostera aseptic seedling, is characterized in that concrete steps are as follows:
(1) choose full complete, the grass wrack seeds that seed coat presents brown of form, under the gnotobasis of superclean bench, be placed in sterilized glass container, rinse 3-5 time with sterilizing natural sea-water, water is blotted with sterilizing filter paper;
(2) being 75% ethanolic solution by concentration soaks seed after 30 seconds, then with sterilizing natural sea-water flushing 3-5 time, water is blotted with sterilizing filter paper;
(3) grass wrack seeds being moved to concentration is to soak 20 minutes in 10% hydrogenperoxide steam generator, then uses sterilizing seawater flushing 3-5 time;
(4) seed of sterilizing is disperseed to be inoculated on solid culture medium;
(5) put into constant temperature illumination box and cultivate, condition of culture is: temperature keeps 18 DEG C, intensity of illumination 1500lux, illumination: dark=12h:12h;
(6) observe by sky, find that there is polluter and immediately uncontaminated person is transferred in new medium;
(7), when seedling grows to 1 ~ 2cm length, can be proceeded in corresponding liquid nutrient medium and cultivate, or directly Qu Qi linked groups organizes cultivation.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106069193A (en) * | 2016-06-24 | 2016-11-09 | 何建清 | A kind of method that Tricholoma matsutake sporophore is isolated and purified |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101904304A (en) * | 2010-07-16 | 2010-12-08 | 中国海洋大学 | Cultivating method of zostera aseptic seedlings |
| CN102577699A (en) * | 2012-01-01 | 2012-07-18 | 山东东方海洋科技股份有限公司 | Method for cultivating zostera marina aseptic seedlings |
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101904304A (en) * | 2010-07-16 | 2010-12-08 | 中国海洋大学 | Cultivating method of zostera aseptic seedlings |
| CN102577699A (en) * | 2012-01-01 | 2012-07-18 | 山东东方海洋科技股份有限公司 | Method for cultivating zostera marina aseptic seedlings |
Non-Patent Citations (2)
| Title |
|---|
| 刘延岭等: "大叶藻组织培养中外植体消毒方法的研究", 《农学学报》, vol. 3, no. 3, 20 March 2013 (2013-03-20), pages 54 - 56 * |
| 王素华: "单细胞藻类的固体培养", 《生物学通报》, no. 2, 31 December 1965 (1965-12-31), pages 18 - 1 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106069193A (en) * | 2016-06-24 | 2016-11-09 | 何建清 | A kind of method that Tricholoma matsutake sporophore is isolated and purified |
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Application publication date: 20141105 |