CN1287657C - Improved porphyra haitanensis variety selection and cultivation method - Google Patents
Improved porphyra haitanensis variety selection and cultivation method Download PDFInfo
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Abstract
The present invention relates to selection of good porphyra haitanensis varieties by utilizing artificial mutation, somatic cell clone, parthenita, etc., and belongs to alga cultivation. Variation cells of the porphyra haitanensis are obtained by a chemical artificial mutation method or a physical artificial mutation method, are singly separated by enzyme of biologic tools, and are cultured to complete thalli. According to the indexes of the protein content amounts of main photosynthetic pigments and pigments of thethalli, the ratio of the main photosynthetic pigments to the pigments, the free amino acid content amount, the growth speed, the thickness and the color of fronds, etc., the good mutant thalli are filtered out, and carry out enzymolysis again to obtain isolation somatic cells to carry out regeneration culture. The prepared infant thalli are singly separated, and are cultured to large thalli, and thus, filaments with pure line are prepared through parthenita. The good properties of F1 thalli of the filaments with pure line and good variety carry out genetic stability analysis and comprehensive quality measurement, the filaments with pure line and good variety are enlarged and cultured and are transplanted into shells to become shell filaments, and then the shell filaments carry out field seedling collection and culture tests. The filaments have good variety and stable property after being proved by the field test, are stored for a long term in a free filament mode, and are popularized and applied. Compared with the existing field quality seed selection techniques of the laver, the present invention has the advantages of high quality variety selection speed, wide selection range, pure variety, stable and good property, convenient storage and use of good variety, etc.
Description
Technical field
The present invention relates to utilize technology seed selection Porphyra haitanensis breedings such as induced mutations, somatic cell clone and unisexual reproduction, belong to the cultivation of marine alga.
Background technology
Laver is the very high alga food of a kind of nutritive value, as ocean health food, is subjected to liking of China, Japan, Korea and the Southeast Asian countries people deeply.The cost of laver culture is low, the cycle is short, the output value is high, is the coastal very important mariculture industry of China.The porphyra yezoensis that the southern Porphyra haitanensis and the north are arranged by the laver of large-scale farming in China.80% of China's laver output is Porphyra haitanensis, and porphyra yezoensis accounts for 20%.Closely during the last ten years, the porphyra yezoensis of China is cultured owing to introduced the good breed variety and the advanced machining technology of Japan, and the output and the output value have obtained significant progress, and most product is sold to the overseas market.Though the annual production of Porphyra haitanensis accounts for about eighty per cant of national laver output, its output value is not as good as porphyra yezoensis.So it is very urgent to the economic benefit and the expansion breed scale that improve China's Porphyra haitanensis aquaculture to cultivate the breeding that is fit to cultivation.
The laver breeding is mainly carried out in Japan and China.Japan has carried out a large amount of fine-variety breeding work to main breed kind porphyra yezoensis.In the sixties in last century, Japan has carried out land for growing field crops (sea area) the fine-variety breeding work of porphyra yezoensis, and successfully must select the altricial ripe improved seeds of slender type; The seventies, new varieties are promoted in the whole nation, make the annual production quantum leap; Simultaneously, they isolate nature pigment mutant again from laver culture colony.To the eighties mid-term, obtained breeding by the mutual cross of difference nature pigment mutant phase.Early eighties, China has tentatively carried out the land for growing field crops breed breeding of porphyra yezoensis; On the other hand, the porphyra yezoensis production of China begins to introduce fund, excellent strain and the advanced machining technology of Japan, and the output and the output value were stepped on a new stage in 10 years.Porphyra haitanensis is the peculiar kind of China, has individuality greatly, and growth is fast, characteristics such as output height.Though, basic biology, the history of life, complete artificial collection of seedling, the free conchocelis of Porphyra haitanensis are collected seedling and cultural technique has been carried out number of research projects (Fujian Province marine products bureau, Porphyra haitanensis is propagated artificially), 1979, the Fujian People's Press); But the breed breeding work of Porphyra haitanensis was not almost carried out, and produced the breed variety that upward uses at present and still was wild species.
Can be divided into miniature filamentous and large-scale two stages of thallus the history of life of Porphyra haitanensis, its thallus is by large-scale farming and to be made into commodity edible.The nuclear phase of filamentous is diploid generation (2n), and through several growth and development stages, final maturation is emitted conchospore (2n), and conchospore develops into haploid thallus (n) after sprouting.Exhausted most thallus is gonochorism, and after cutting one's eye-teeth, male thallus produces the anthreid device, the sperm that diffuses (n) with water waft to and attached on the female thallus, the carpogonium ripe (n) fertilization with it.The carpogonium (2n) of fertilization develops into the carpogonium ascus, and the carpospore that maturation diffuses out (2n) pierces shell or develop into filamentous (2n) in seawater.Porphyra haitanensis thallus can not be diffused single spore and be carried out vegetative propagation.(volumes such as Ceng Chengkui, Wang Sujuan, Liu Sijian, 1985.Algal cultivation is learned, Science and Technology of Shanghai publishing house).Because Porphyra haitanensis thallus is gonochorism, does not have single spore vegetative propagation approach again.In addition, its reductional cell division occurs in conchospore on earth diffuse before or diffuse after the sprouting stage do not make clear, genetic breeding work is very difficult.We separate the thallophytic single cell of Porphyra haitanensis and cultivate, the thallus cell differentiation, and basic genetic research (Wang Sujuan, Zhang Xiaoping, the Xu Yunlong etc. of more than ten years have been carried out in mutant separation and hybridization etc." research that Porphyra haitanensis trophozyte and protoplast are cultivated ", Oceanologia et Limnologia Sinica .1986,17 (3): 217-221; Yah X H, Wang S J.Studies on the development and differentiationof the somatic cells from Porphyra spp. (Rhodophyta) .Marine Sciences.1990.3 (2): 195-208; Yan Xinghong, 2003. country's 863 great special research projects " Porphyra haitanensis fine-variety breeding technology " achievement Report in mid-term), it is the same with hermaphroditic porphyra yezoensis to have illustrated Porphyra haitanensis, and reduction division occurs in first and second cell divisions after the conchospore sprouting; There is certain difference in 4 cells that postmeiotic produces in heredity, these four cells continue division and develop into big thallus.So a Porphyra haitanensis thallus of being come by the conchospore growth belongs to chimera in heredity.
The somatic cell separating tool enzyme of laver, the production application of trophozyte seed, pure laver line cultivation etc. has many research reports to see also separating of Tang Yanlin laver nutritive cell and protoplast and cultivation (Shandong Marine College's journal, 1982,12 (4): 37-50); Fang Zongxi, Dai Jixun, the enzyme process that lavers such as Tang Yanlin are thin separates and the application in aquaculture (Marine Sciences, 1986,10 (3): 46-47; The research that Porphyra haitanensis trophozyte such as Wang Sujuan, Zhang Xiaoping, Xu Yunlong and protoplast are cultivated) (Oceanologia et Limnologia Sinica .1986,17 (3): 217-221), and relevant patent report, China Patent No. is respectively 85104059,91107320,96116039 and 01132162.But Porphyra haitanensis fine-variety breeding technology there is not patent report both at home and abroad.
Purpose of the present invention: utilize modern biotechnology, set up a cover effectively, fast, Porphyra haitanensis fine-variety breeding technology is applied to produce so that select the Porphyra haitanensis breeding reliably, improves the economic benefit and the social benefit of China's laver culture industry.
Summary of the invention
The seed selection step is:
(1) diffuses carpospore with the wild Porphyra haitanensis thallus of nature, make its germination and growth, to obtain the wild species free conchocelis;
(2) diffuse out conchospore from the wild species free conchocelis, and it is cultivated into thallus, with artificial mutagen they are carried out mutagenic treatment then, cultivate again;
(3) handle thallus after mutagenesis with the marine bacteria enzyme, isolate single from cell and protoplast, and they are cultivated into thallus;
(4) select index to select good regeneration thallus according to breeding, and carry out individual plant and cultivate;
(5) to being undertaken quantitatively and qualitative determination by the good thallophytic main photosynthetic pigment selected and chromoprotein and live body absorption spectrum, good and bad and stable with proterties such as the growth of judging laver and qualities;
(6) the good thallus of enzymolysis and is cultivated into regeneration plant to them obtaining single cell and protoplast once more, utilizes the unisexual reproduction technology, obtains diplontic pure lines filamentous;
(7) after pure lines filamentous maturation, make it emit conchospore, cultivate into F
1For thallus, and it is carried out the check of attributional analysis and genetic stability;
(8) transplant in shell after the free conchocelis pulverizing with excellent strain, cultivate into shell conchocelis, then, adopt conchospore in culturing on the lace curtaining, carry out the breeding production test of thallus sea area from shell conchocelis;
(9) breeding remains on indoor with the free conchocelis form.
Described induced mutations agent is N-methyl-N '-nitro-N-nitrosoguanidine (MNNG);
Described induced mutations agent also
60The Co-gamma-rays;
The derived bacterium that comes of described marine bacteria enzyme is false pseudomonas bacillus (Psuedomonas sp);
Described main photosynthetic pigment and chromoprotein are: chlorophyll a, phycoerythrin and phycocyanin.
Compare with existing land for growing field crops laver fine-variety breeding technology, the present invention has following advantage: (1) strain breeding is wide, and obtainable excellent strain amount is many.Owing to used the induced mutations technology, mutant is increased on quantity and type significantly, in their cytothesis body, can obtain the variant of the different merits of big measurer, be used for further breeding and select.(2) breeding variety speed is fast.Owing to utilized the somatic cell fast breeding technique, the thallus cultivation and the excellent strain that can carry out for several times at indoor 1 year are selected, season limit overcome in the laver fine-variety breeding of land for growing field crops owing to can only obtain once thallophytic weak point in 1 year, but thereby obtain within a short period of time (2-3) to be produced the excellent strain that uses, and traditional land for growing field crops fine-variety breeding method efficient is low, the cycle long (more than 5 years).(3) breeding of Huo Deing is the genotype pure lines, and merit is very stable.In view of the reduction division of Porphyra haitanensis occurs in first and second cell divisions after conchospore is sprouted; There is certain difference in 4 cells that postmeiotic produces in heredity, these 4 cells continue division and develop into the hereditary chimeric big thallus that upward belongs to.The present invention breeds by the continuous quadratic somatic cell clone and separates excellent strain, has guaranteed that the product that obtain are the pure lines on the real hereditary meaning.The breeding pure lines that are separated to are because its allelomorph of filamentous (dliploid) isozygotys fully, so they thallus (monoploid) from generation to generation in, the individual inheritance background is identical, in identical growing environment, it is very little that individual difference becomes, the separation of merit can not occur, guarantee the output and the quality of breeding.The bar class laver breeding that obtains with traditional land for growing field crops fine-variety breeding technology owing to often be not pure lines, filial generation thallus from generation to generation in, the separation of merit often occurs and lose, need one side to use one side to select again, otherwise, the difficult merit of keeping breeding.(4) its effect of separating laver individual cells and protoplast of the marine bacteria enzyme of the present invention's use is good more than the sea snail enzymes that uses on the market, and enzyme pair cell toxicity is low, isolated cells survival rate height, guaranteed to be separated effectively and cultivate into plant, accelerated the screening of breeding through the mutant that induced mutations obtains.
Embodiment
1. gather the wild Porphyra haitanensis thallus of self-sow, behind the removal of impurities algae, clean up, and airing, dehydration about 70%.Again thallus is soaked in the sterilization seawater, collects the carpospore of diffusing, and they are cultivated into free conchocelis by thallus.
2. diffuse conchospore from the wild species free conchocelis, and it is cultivated into thallus, then they are carried out induced mutations and handle.Mutagen are selected MNNG for use, and concentration of treatment is 10-25ppm, and processing time 30min also can use
60The Co-gamma-rays is handled, and exposure dose is 500-1500Gy.After treated thallus qi of chong channel ascending adversely cultivated for 2 weeks, be used for enzymolysis, isolated cell.
3. the thallus that is used for isolated cell, after sterilization seawater cleaning 3-5 time, (2mm cuts into pieces
2About), drop into enzymolysis in the enzyme liquid.Enzyme liquid contains 0.5% marine bacteria enzyme, 0.8M mannitol, 60% nature seawater and 40% distilled water.Enzymatic hydrolysis condition: 26 ℃, 1.5h, dark.Then, with 200 purpose bolting silks single from cell and protoplast filter out, with centrifugal process (1000rpm, 5min) earlier enzyme liquid is removed, be 1.035 seawater suspension cell and centrifugal once more (1000rpm with proportion again, 5min), after continuous centrifugal washs 3 times successively, the gained cell is put the MES medium carry out liquid culture.
4. the cultivation of single cell and protoplast and plant regeneration condition: 20 ℃, the light intensity in the 1st week is 1000lux (12L:12D), and after the 2nd week, light intensity increases to 3000lux (12L:12D).After cell development becomes little thallus, they are moved to qi of chong channel ascending adversely cultivation the blake bottle in culture dish, temperature-resistant, light intensity increases to 5000lux (12L:12D) simultaneously.When regeneration thallus body reaches 1cm, select the variant of tool merit, carry out single cultivation.In addition, the good regeneration thallus of being selected is carried out following main photosynthetic pigment and chromoprotein----chlorophyll a, the assay of phycoerythrin and phycocyanin is because the content of these three kinds of pigments and albumen height and the ratio between them have determined quality and the growth of laver; Simultaneously, measure the live body absorption spectrum, measure the content of free amino acid and gross protein, measure the thickness and the gloss of frond.
5. the good regeneration thallus of being selected when quilt reaches certain size, goes out single cell according to the said method enzymolysis once more, and they are cultivated into regeneration thallus.When the fast maturation of the good thallus of cultivating from little individual plant, utilize unisexual reproduction, obtain diplontic filamentous pure lines.
6. after pure lines filamentous maturation, make it emit conchospore, cultivate into F
1For thallus, and it is carried out the check of attributional analysis and genetic stability.
7. the free conchocelis of the excellent strain that proterties is stable is pulverized, and transplants in shell, cultivates into shell conchocelis; Then, adopt conchospore in culturing on the lace curtaining, carry out sea area thallus breeding production test with shell conchocelis; Thallus to mariculture is grown once more, quality, and comprehensive analysis such as disease resistance are selected excellent strain.
8. the breeding free conchocelis that obtains is cultivated at 15 ℃, preserved under 300lux (12L:12D) condition.
Claims (7)
1, Porphyra haitanensis fine-variety breeding method is characterized in that: the seed selection step is:
(1) diffuses carpospore with the wild Porphyra haitanensis thallus of nature, make its germination and growth, to obtain the wild species free conchocelis;
(2) diffuse out conchospore from the wild species free conchocelis, and it is cultivated into thallus, with artificial mutagen they are carried out mutagenic treatment then, cultivate again;
(3) handle thallus after mutagenesis with the marine bacteria enzyme, isolate single from cell and protoplast, and they are cultivated into thallus;
(4) select index to select good regeneration thallus according to breeding, and carry out individual plant and cultivate;
(5) to being undertaken quantitatively and qualitative determination by the good thallophytic main photosynthetic pigment selected and chromoprotein and live body absorption spectrum, good and bad and stable with proterties such as the growth of judging laver and qualities;
(6) the good thallus of enzymolysis and is cultivated into regeneration plant to them obtaining single cell and protoplast once more, utilizes the unisexual reproduction technology, obtains diplontic pure lines filamentous;
(7) after pure lines filamentous maturation, make it emit conchospore, cultivate into F1, and it is carried out attributional analysis and genetic stability check for thallus;
(8) transplant in shell after the free conchocelis pulverizing with excellent strain, cultivate into shell conchocelis, then, adopt conchospore in culturing on the lace curtaining, carry out the breeding production test of thallus sea area from shell conchocelis;
(9) breeding remains on indoor with the free conchocelis form.
2, according to the Porphyra haitanensis fine-variety breeding method of claim 1, it is characterized in that: described induced mutations agent is N-methyl-N '-nitro-N-nitrosoguanidine.
3, Porphyra haitanensis fine-variety breeding method according to claim 1 is characterized in that: described induced mutations agent is 60Co-gamma-rays also.
4, Porphyra haitanensis fine-variety breeding method according to claim 1 is characterized in that: the derived bacterium that comes of described marine bacteria enzyme is false pseudomonas bacillus (Psuedomonas sp).
5, Porphyra haitanensis fine-variety breeding method according to claim 1 is characterized in that: described main photosynthetic pigment and chromoprotein are: chlorophyll a, phycoerythrin and phycocyanin.
6, Porphyra haitanensis fine-variety breeding method according to claim 1 is characterized in that: the good thallophytic leading indicator of described choice: thallophytic build, color, growth rate and sooner or later ripe.
7, Porphyra haitanensis fine-variety breeding method according to claim 1 is characterized in that: described attributional analysis content is: the color and luster of free aminoacid content, total protein content, frond thickness and dry product.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN101167439B (en) * | 2007-11-16 | 2010-04-21 | 中国海洋大学 | Polysiphonia urceolata tetraspore seedling formation method |
| CN102334443A (en) * | 2010-07-15 | 2012-02-01 | 国家海洋局第三海洋研究所 | Method for obtaining porphyra haitanensis filament-shaped algae with high content of phycoerythrin by optimizing culture control condition |
| CN102334444A (en) * | 2010-07-27 | 2012-02-01 | 国家海洋局第三海洋研究所 | Method for improving growth rate of porphyra haitanensis filamentous algae by adopting optimized regulation culture conditions |
| CN103340151A (en) * | 2013-07-15 | 2013-10-09 | 上海海洋大学 | Screening method of low salt-resistant fine-strain porphyra haitanensis |
| CN105379613A (en) * | 2015-11-28 | 2016-03-09 | 王谷安 | Laver culturing rope capable of preventing laver culturing raft frame from being attached with hybrid algae |
| CN106857237A (en) * | 2017-03-01 | 2017-06-20 | 集美大学 | A kind of method of porphyra haitanensis Doubled haploid breeding |
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