CN1138466C - Method for culturing pure laver line - Google Patents

Abstract

The present invention relates to the technical field of seed culture and production of laver cells, and provides a method for culturing a pure laver line. Immature laver leaves are decomposed by ocean phytophagy shellfish protein extracting solution, and a single cell or a protoplast is generated. Then, a chromosome is cultured or induced to increase times, and thus, a diploid filament pure line is generated. The cultured thalli of a hermaphroditic type are singly cultured to be mature and to be automatically impregnated, and thus, a pure diploid filament is generated; therefore, the present invention carries out seed protection or seed production. A completely pure laver filament of a hermaphroditic type or a dioecious type is prepared by the direct time increase of the chromosome, is amplified to be cultured, and is used for protecting seeds or producing seeds. The present invention can select the individual with good property to culture pure laver line for materials, all good properties of the parents are completely maintained due to no separation of filial generations, and thus, the present invention can largely enhance the output and the quality of the laver in cultivation, and is popularized and used for culturing laver seeds.

Description

A kind of method for culturing pure laver line

Technical field

The present invention relates to a kind of laver cell and carry out the seed rearing production technical field.

Background technology

Laver makes it to become one of China's two Large Marine Ecosystem algae industries because of nutritious and great potential medical value, output row third place in the world.The gross output value of China laver in 2000 reaches about 200,000,000 yuan.In China, before the history of people's edible laver can be traced back to more than 1,000 year, laver aquaculture history also had centuries.In recent decades, along with the development of laver cultivation industry, people are more and more higher to the requirement of laver variety and quality.But up to the present, also do not cultivate the report of the pure lines laver with merit and energy genetic stability.This present situation is unbecoming with its industry size, and is also totally unfavorable to the development of laver cultivation industry.So set up laver germplasm storehouse, cultivate pure lines laver with merit and energy genetic stability, be very crucial for promotion laver cultivation industry health, sustainable development.

Laver history of life by gametophytic generation--thallus (n) and sporophyte generation--filamentous (2n) is formed.Produce gamogenesis organ carpogonium (n) and anthreid device after the thallus maturation.Make the fertilization of carpogonium cell after sperm (n) diffuses, become fertilization carpogonium (2n), form carpospore through mitosis.Carpospore disengaging frond pierces in the stone matter matrix and (mostly is shell under the natural conditions), sprouts into filamentous.Filamentous is the summer form of crossing of laver.Filamentous is grown a period of time, produces conchospore (n) through reduction division.The autumn air temperature rapid drawdown, conchospore diffuses, and is attached on nature or the artificial growth substrate to sprout into thallus (n) seedling.Except that sexual reproduction, laver also has single spore, does not have and join multiple asexual reproduction modes such as spore, endospore, sees also volumes such as once being Kui, Wang Sujuan, Liu Sijian and annotates " algal cultivation " (Science and Technology of Shanghai publishing house, 1985).

We and other researchers show that to genetics and the molecular biology research that laver carries out the genetic diversity of laver is very abundant simultaneously, and the genetic diversity of cultivated population does not significantly reduce yet.Our experimental data shows that further the allelomorph of laver is very abundant, " the RAPD labeling technique detects the application close in the Germplasm Identification in the laver diversity " (Botany Gazette of writing referring to Jia Jianhang, Wang Ping, Jin Minde etc., 2000,42 (4): 403-407).Though the hypoploidy (n=2 to 7) of laver, the reorganization combination is few, because the allelomorph number is big, so the probability of equipotential genetic heterozygosis is very high in the colony, genetic diversity is abundant, and individual difference is big.This phenomenon is very disadvantageous to production practices.

Recently the biotechnology kind is that breeding method generally is to collect carpospore ripe on the blade, makes it be grown to filamentous, and gets up as kind of a reservation with this.Though do not get rid of the autogamous possibility of reproductive cell on the thallus, in most cases, the sperm that makes the fertilization of carpogonium cell is from the Different Individual in the cultivated population.Though so coexist on the blade as fertilization result's carpospore, actual is the result of colony's natural insemination.The filamentous strain that obtains like this is actual to be the set of a colony.Even the filamentous (2n) that forms by single carpospore cell development, because of its homologous chromosome many from different parent's individualities, so be heterozygosis.Because the probability of equipotential genetic heterozygosis is very high in the colony, when reduction division forms conchospore (n), Mendel will be taken place separate.

The nineties, someone utilizes the laver nutritive cell to carry out seed production, referring to Fang Zongxi, Dai Jixun, Tang Yanlin etc. " enzyme process of laver cell separates the application that is combined in the aquaculture " (Marine Sciences, 1986,10 (3): 46-47) relevant patent report with it, China Patent No. are respectively 85104059 and 96116039.X, but it is only from the trophosome to the trophosome, monoploid is to haploid process, the cultivation that is not sheerly; Owing to be difficult to control allogamous generation in producing, isozygoty so can not guarantee the kind system on the genetics meaning.Certainly, the scholar that also has proposes to get the thallus of porphyra radical leaves and is divided into small pieces and cultivates filamentous and carry out strain development.But this method can not be got rid of the existence of chimeric cell in the zonule, so still face the impure problem of strain.

Summary of the invention

Technical problem to be solved by this invention is to provide a kind of method for culturing pure laver line at prior art, the filamentous that it obtains, its two covers homologous chromosome is in full accord, allelomorph isozygotys fully, proterties can not separated in the breeding afterwards, therefore has the pure lines on real genetics and the thremmatology meaning.

The present invention solves the problems of the technologies described above the technical scheme that is adopted: this main laver pure lines breeding method is characterized in that step is: 1) choose immature laver blade, and remove assorted algae and antibiotic treatment; 2) utilize ocean phytophagy shellfish protein extract to decompose blade, obtain the unicellular or protoplast of laver blade; 3) separate unicellular or protoplast is cultivated, make them generate new blade; Induce its chromosome to increase naturally doubly on the basis of perhaps unicellular or protoplast, generate the dliploid filamentous that isozygotys and be sheerly in single thallus doubly; 4) for hermaphroditic kind, can be cultured to sexual maturity separately with cultivating the thallus of coming out, make its self-fertilization, produce two times of filamentouss that isozygoty, thereby protect the production of kind or seed; For hermaphroditism or dioecious kind, increase the conchocelis of porphyra that isozygotys fully that doubly directly obtains through chromosome and carry out amplification culture, be used for protecting and plant or seed production.

Above-mentioned ocean phytophagy shellfish is winkle, Rong spiral shell or oyster or abalone.

Compared with prior art, the invention has the advantages that: the technology of the present invention is based upon on the unicellular vegetative basis, with the somatic cell (n) without Sex Differentiation is material, isolates unicellular or protoplast, and directly induced chromosome doubles naturally and forms filamentous (2n); Perhaps carry out vegetative propagation and form new thallus (n), control hermaphroditic laver and carry out self-fertilization, further develop into filamentous (2n).The condition induced chromosome of utilization doubles the filamentous with two kinds of approach acquisitions of self-fertilization naturally, and its two covers homologous chromosome is in full accord, and allelomorph isozygotys fully, so proterties can not separated in the breeding afterwards.Therefore this method is cultivated is pure lines on real genetics and the thremmatology meaning.

Non-pure lines laver is owing to allelomorph in the colony is abundant, so hereditary difference is big, individual difference is obvious.The pure lines laver is because allelomorph isozygotys fully, and the individual inheritance background is in full accord, and under the identical situation of planting environment, individual difference will be very little.Selecting the good individuality of individual proterties is the pure lines laver that material is cultivated, because separating does not appear in filial generation, all merits that keep the parent fully, so the cultivation laver can be in output and can be greatly improved qualitatively, economic benefit and social benefit are fairly obvious.

Embodiment

Below embodiments of the invention are described in further detail.

1, chooses fresh immature laver blade; Or the laver blade of marginal portion maturation, margins of excision keeps prematurity zone, center as far as possible.Remove assorted algae, handled respectively 10 minutes, repeatedly wash in order to sterile working with the sterilization seawater then and use with 0.7%KI and 1% penicillin.The blade central area of handling well is cut into 1mm ²About small pieces.

2, utilize intertidal zone, ocean phytophagy shellfish such as winkle, Rong spiral shell, oyster or the like protein extract, its concentration 1%, other adds NaCl or glucose, makes its final concentration be respectively NaCl 1.5mol/L or glucose 2mol/L, with the 1mm of 1 step processing ²Small pieces carry out enzymolysis in 20 ℃, note observing the enzymolysis situation, are that available sterilization seawater repeatedly washes after the peripheral 5-6 confluent monolayer cells of blade is loosening, wash enzyme liquid off, and filter with 300 mesh sieve thin,tough silk, the unicellular or protoplast of acquisition laver blade.

3, separate unicellular or protoplast carries out cell culture.Cell culture is carried out in the space of illumination box or other controllable condition.Culture fluid is the seawater of Ensure Liquid salt, it contains N-4ppm, P-0.4ppm, beginning 4-5 days condition of culture is 20 ℃, 1500LUX, photoperiod is L: D=12: 12, when condition of culture changes about 15 ℃ 1500LUX into, photoperiod is L: D=10: 14, and unicellular or protoplast can generate new blade; Perhaps change into about 20 ℃ when condition, 1500LUX, the photoperiod is L: D=14: induce its spontaneously doubled haploid on the basis of 10 or protoplasts unicellular in single thallus doubly, generate two times of filamentouss pure lines of isozygotying.

4,1 for hermaphroditism or dioecious kind, all can be directly with the conchocelis of porphyra that isozygotys fully that obtains, and carry out amplification culture, be used for protecting kind or seed produces.And different, for example the porphyra yezoensis filamentous is 19 ℃ to concrete amplification cultivation condition because of the kind difference, 1500LUX, and the photoperiod is L: D=12: 12, the Porphyra haitanensis filamentous is 21 ℃, 1500LUX, the photoperiod is L: D=12: 12.

4.2 for hermaphroditic kind, also the thallus that said method can be turned out is cultured to sexual maturity separately, makes it carry out self-fertilization, forms carpospore, carpospore is diffused, and produces two times of filamentouss that isozygoty, and plants or seed production thereby protect.

Claims (2)

1, a kind of method for culturing pure laver line is characterized in that step is:

1) chooses immature laver blade, and remove assorted algae and antibiotic treatment;

2) utilize ocean phytophagy shellfish protein extract to decompose blade, obtain the unicellular or protoplast of laver blade;

3) separate unicellular or protoplast is cultivated, make them generate new blade; Induce its chromosome to increase naturally doubly on the basis of perhaps unicellular or protoplast, generate the dliploid filamentous that isozygotys and be sheerly in single thallus doubly;

4) for hermaphroditic kind, can be cultured to sexual maturity separately with cultivating the thallus of coming out, make its self-fertilization, produce two times of filamentouss that isozygoty, thereby protect the production of kind or seed; For hermaphroditism or dioecious kind, increase the conchocelis of porphyra that isozygotys fully that doubly directly obtains through chromosome and carry out amplification culture, be used for protecting and plant or seed production.

2, method according to claim 1 is characterized in that described ocean phytophagy shellfish is winkle, Rong spiral shell or oyster or abalone.