CN1258969C - Porphyra conchocelis pure line making method - Google Patents

Abstract

The present invention relates to a method for producing seeds of hemiphyll laver filament pure lines in north china. The present invention is characterized in that individuals with different sexual characteristics are selected out of a wild population; the individuals are scrubbed by absorbent cotton balls and are pasted on a plane, and excessive sea water is drained; an algae piece of 1 to 2mm is cut from the hermaphroditic alga base near the paroecious boundary line position; the alga pieces with the same size are cut from the middle base of the alga for the unisexual individuals; all cells are determined to be nutrient cells; the cells are respectively cleaned by ultrasonic; the cells are cultured under the conditions of the temperature of 15 to 20 DEG C, the optical intensity of 1000 to 6000 lux, and the illumination time of 10 to 14; a filament alga group is grown after 1 to 3 months; the single alga group is separated to be cultured and enlarged. The method has the advantages that the obtained filament is a pure line and is suitable for individuals with different sexual characteristics; two filament pure lines can be obtained from one hermaphroditic individual; the contamination organisms are removed by ultrasonic, and thus, the success ratio is high; the present invention possibly has common adaptability in laver types with same sexual characteristic.

Description

The producing method for seed of North China half leaf conchocelis of porphyra pure lines

Technical field

The present invention relates to the producing method for seed of a kind of North China half leaf conchocelis of porphyra pure lines.

Background technology

Laver is important economical alga, but has only a few species by artificial cultivation, and mainly concentrates on country in Southeast Asia as China, Japan and Korea S.North China half leaf laver is the distinctive laver kind in the yellow intertidal zone, the Bohai Sea of northern China, does not become the cultivation kind at present as yet, but because its frond is vivid aubergine or bronzing, compares with the kind that China is common, may contain more phycobniliprotein; And its frond good luster, anti-adhering to, the season of growth can be carried out the artificial breeding cultivation in the lower area of temperature, so higher potential economic worth is arranged in evening winter and early spring.

The thallus of laver is the gametophyte stage, and the germplasm that is easy to preserve is the thread sporophyte stage.The preparation method and the thallophytic sex arrangement mode of pure lines filament germplasm are closely related.It is not single that the sex of North China half leaf thallus of porphyra wild stocks is formed, and this has just determined the pure lines filamentous method that obtains various sex individualities to be not quite similar.Its population compositing characteristic be most individual (accounting for 97%) for hermaphroditism and male and female organize respectively account for the thallus frond does not wait one side of something, that is to say that these individualities are the biological properties that the hermaphroditism kind but has gonochorism kind (as Porphyra haitanensis) on sex.The different sexes tissue district of different with mentioned kind is frond can tell early stage when not forming as yet (be organ of multiplication) that frond is grown; In addition, each individuality of 1.5% being arranged approximately is complete female individuality and complete male individual.

At present, obtain this main laver filament germplasm two approach are generally arranged: one is to obtain filamentous by the carpospore of gathering on the wild frond at indoor cultivation; Another is to cut male and female vegetative tissue respectively, and hybridization formed the hybrid carpospore after the two was cultivated altogether and makes it maturation then, and separation obtains filamentous after sprouting; And according to present result of study, male and female half leaf of North China half leaf laver may originate from different parents respectively.More than the common trait of two kinds of methods be whether filamentous that can not determine acquisition is the pure lines in the heredity, and can't obtain from complete male individual filament germplasm.

In view of cultivation prospect and the production of North China half leaf laver are gone up the more and more higher requirement in blastogenesis source, and the corresponding needs of carrying out North China half leaf laver genetics research, the producing method for seed of setting up the filamentous pure lines is very important.

Summary of the invention

The object of the invention provides a kind of producing method for seed that obtains North China half leaf conchocelis of porphyra pure lines, and it can overcome the above-mentioned shortcoming of prior art, obtains the pure lines in the heredity, satisfies following production and research to purebred demand.

The producing method for seed of a kind of North China half leaf conchocelis of porphyra pure lines is characterized in that selecting the thallus individuality earlier from the wild stocks of North China half leaf laver, and is divided into different groups by sex character; Selected frond is scrubbed with rayon balls in seawater; The frond of washing is attached on the smooth clean plane, and drop falls excess water; Downcut the square algae sheet of 1～2mm at hermaphroditic frond base portion near the marginal position of male and female; If complete female or complete male individual, then downcut onesize algae sheet in position near base portion in the frond; Determine that at test under microscope whole cells all are trophozytes; To clean with ultrasonic respectively from the algae sheet of different groups, microscopy determines that the two sides of algae sheet all do not have assorted algae or protozoa and adhere to then; Cultivate under 15～20 ℃, light intensity 1000～6000lux, 10～14 hours condition of light application time handling clean a slice algae sheet; To there be red filamentous algae to fall to growing after 1～3 month; Single algae is fallen to telling to cultivate amplification.

Advantage of the present invention: 1, used starting material is very little, and the whole operation process does not have the introducing of foreign heredity substance, can guarantee that the genetic origin of the filamentous that obtains is single; 2, owing to existing hermaphroditism individuality in the half leaf laver colony of North China, complete female and complete male individuality is arranged again, the method all is suitable for the individuality of different sexes feature; 3, can obtain the filamentous pure lines respectively by female vegetative tissue and male vegetative tissue from the hermaphroditic individuality of a strain, promptly obtain 2 filamentous pure lines from a strain algae, their ecological characteristic has difference, shows its genetic origin difference; 4, adopt ultrasonic to remove the assorted algae and the protozoa on surface, success rate reaches more than 95% 5, by checking, this method has general applicability in having the laver kind of identical sex character, as, kind Porphyra purpurea thallophytic sex in the Atlantic Ocean, North America is arranged similar to North China half leaf laver to the population composition, can use the same method to obtain the filamentous pure lines.

Embodiment

The inventor takes back the laboratory with the North China half leaf thallus of porphyra individuality that picks up from intertidal zone, peaceful angle, Qingdao spring calendar year 2001, chooses the individuality of hermaphroditism, complete female and full hero through preliminary go-on-go; Select individuality respectively, in filtering sea, do preliminary cleaning,, generally need to clean 2～3 times to remove the main aufwuch in frond surface with rayon balls with purpose proterties; The frond that to choose launches in paper or culture dish and drop falls redundant moisture, does because North China half leaf laver is anti-, is noted that unsuitable long in order to avoid frond cell of operating time especially because excessively dehydration and death; Or when frond will be done, the clean seawater that takes a morsel with dropper dripped on the frond; Downcut vegetative tissue fritter (general 1～2mm is square) with clean blade then, to hermaphroditism type individuality, chosen position generally is the close marginal position of male and female of the base portion of frond; To complete female or complete male individual, generally choose position near the frond base portion; Microscopy determines that the algae sheet that cuts all is made up of trophozyte; The algae sheet that will partly be obtained by the single sex of individual plant frond is placed in the small beaker of the centrifuge tube of 10ml or 50ml, adds the seawater (for 1/2～2/3 height of container) that boils the back cooling, with ultrasonic crusher mix algae and protozoic removing; That the inventor uses is the ultrasonic crusher VC750 of U.S. SONICS--MAREIALS company, and using amplitude is 35%; Because the frond of North China half leaf laver is thin, handle about 5 seconds in order to avoid the overlong time pair cell damages at every turn, repeats 3 times, change the water cleaning at every turn.After handling, the two sides of microscopy algae sheet does not all have assorted algae and protozoa and pollutes and promptly to achieve the goal, if still have assorted algae, can repeat above ultrasonic treatment step, but in order to guarantee not damage the frond cell, should reduce to some extent each time that continues.Then, every algae sheet is placed on the triangular flask of 100ml or beaker is cultivated, temperature is 18 ℃ of C, and light intensity 1500lux, changed and once cultivates seawater at 12 hours every days of light application time in 2～3 weeks.After 2 months, all there is the filamentous algae to fall to growing on the bottle end and the algae sheet; Single algae is fallen to choosing cultivation separately in the triangular flask of a 250ml, can grow the filamentous of a great deal of after 1～3 month, be purpose product filamentous pure lines from the different sexes frond.

Generally less by the thread scale of construction that the male tissue of hermaphroditism individuality and male entirely individual male tissue obtain at first with method of the present invention, the time of separation can suitably postpone; And generally more by the thread scale of construction of female sex organization acquisition, fall for the ease of obtaining single algae, macroscopic algae falls and will separate as early as possible.

Claims (1)

1, the producing method for seed of a kind of North China half leaf conchocelis of porphyra pure lines is characterized in that selecting the thallus individuality earlier from the wild stocks of North China half leaf laver, and is divided into different groups by sex character; Selected frond is scrubbed with rayon balls in seawater; The frond of washing is attached on the smooth clean plane, and drop falls excess water; Downcut the square algae sheet of 1～2mm at hermaphroditic frond base portion near the marginal position of male and female; If complete female or complete male individual, then downcut onesize algae sheet in position near base portion in the frond; Determine that at test under microscope whole cells all are trophozytes; To clean with ultrasonic respectively from the algae sheet of different groups, microscopy determines that the two sides of algae sheet all do not have assorted algae or protozoa and adhere to then; Cultivate under 15～20 ℃, light intensity 1000～6000lux, 10～14 hours condition of light application time handling clean a slice algae sheet; To there be red filamentous algae to fall to growing after 1～3 month; Single algae is fallen to telling to cultivate amplification.