CN104920201A - Laminaria germplasm storing method and laminaria germplasm seedling cultivating method - Google Patents
Laminaria germplasm storing method and laminaria germplasm seedling cultivating method Download PDFInfo
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Abstract
The invention discloses a laminaria germplasm storing method and a laminaria germplasm seedling cultivating method. The laminaria germplasm storing method includes the steps of preprocessing laminaria daughter spore explant or laminaria macrospore explant, inducing callus, and storing the callus. The laminaria germplasm seedling cultivating method includes the steps of spreading the cultivation of the callus and conducting seedling differentiation of the callus. The germplasm storage and seedling cultivation of the callus of the laminaria explant are not influenced by seasons or weather, the rapid frond reproduction aim can be achieved within a short period of time, productive laminaria seedlings pure in seed nature, good in quality and high in yield can be produced, the methods are not limited by mature spore, growth parts of spore of each growth period can be used as explant to induce the callus, the obtained callus can be used as germplasm material to be stored for a long time, cultivation spreading and differentiation can be further achieved, the methods are used for breeding and seedling producing, and the methods have the advantages of being high in applicability, easy to control manually, low in material consumption, high in efficiency, easy to operate and convenient to popularize.
Description
Technical field
The present invention relates to preserving seed and offspring seed cultivation method thereof, the kelp germplasm particularly related to for breeding production is preserved and offspring seed cultivation method, belongs to marine alga tissue cultures and applied technical field.
Background technology
Sea-tangle is large-scale sea-plant, has larger economic worth.Natural classification system belongs to Phaeophyta, brown sub-guiding principle, Laminariales, Laminariaceae, Larminaria.Sea-tangle is cold water algae, and natural distribution is in the sea area of high latitude.Grow nonparasitically upon another plant on the cay of seabed, individuality is comparatively large, is that artificial cultivation produces maximum marine algas more.Sea-tangle by large-scale sporophyte (i.e. commodity production object " sea-tangle ") from generation to generation and small gametophytic generation is alternate with each other forms throughout one's life.The large individuality of sporophyte is tame object, and the time in the history of life that accounts for is long.Gametophytic generation is the object of artificial culture seed, and present production is all carried out under constant indoor temperature discharge condition.Sea tangle sporophyte is divided into blade, handle and holdfast three parts.The frond blade of growing up in banded, without branch, brown and be rich in gloss.There are two shallow ridges to pass through and form " middle band portion " in the middle part of blade.The place that blade base is connected with handle is the growing part of frond, the sea-tangle of length more than one meter, the position of growing part from blade base to the position apart from base portion 10 cm, the cell division function at this position is very strong.In sea-tangle process of growth, growing part cell constantly divides, new organization is constantly increased from blade base, old tissue is then pushed to front, so from blade base more away from the time of organizing it to be formed also more early, thus aging also early, start at first to degrade, growth between this growth pattern of sea-tangle is just called.The algae of this growth, its meristematic tissue is positioned at the base portion of blade, and in process of growth, new organization constantly increases from leaf base, passes, show the growth of algae to ending, simultaneously taper gradually aging come off.The object of higher plant tissue cultures obtains complete plant, because be support without earth culture, so with solid culture medium to play the effect of fixing plant, and sea-tangle belongs to aquatic sea-plant, can be immersed in liquid nutrient medium completely and cultivate, and liquid nutrient medium nutrient component is even, so inoculum density can be much larger than solid culture medium, such output is also large.
The callus concept of brown alga meets higher plant, and this callus gives appropraite condition, just can again break up, and develops into normal sporophyte; Can also preserve for a long time; Also cultivate by suspending and breed rapidly, carrying out nursery production to be used as clone.The research of brown alga callus starts from saga(Saga, N., T. Uchida & Y. Sakai in 1978,1978.
clone Laminaria from single isolated cell. Bull. jap. Soc. sci. Fish. 44:87.) study sea-tangle, he, after acquisition callus, is separated to individual cells, and induces into sporophyte, demonstrate the totipotency of brown alga callus cell first from callus.Yan Zuomei (marine products institute of Chinese Marine University retirement professor, Fang Zongxi, Yan Zuomei, Wang Zongcheng;
the preliminary observation of sea-tangle and undaria pinnitafida tissue cultures[J]; Science Bulletin; Phase nineteen eighty-two 11) once the different tissues of sea-tangle and undaria pinnitafida was made comparisons in 1984, find the maximum probability that blade base (growing point) and middle band portion form callus and break up, shank takes second place, and rhizoid portion is the poorest, and rear two parts are difficult to be divided into sporophyte again.(1967.3-, Institute of Oceanology of the Chinese Academy of Sciences assistant researcher, Wang Xihua, Qin Song, the Zeng Chengkui such as Wang Xihua;
the high efficiency induction of sea-tangle callus[J]; Oceanologia et Limnologia Sinica; 06 phase in 1999) to utilize solid culture medium to induce sea tangle sporophyte to obtain callus in 1999, but have no the spreading cultivation of callus, break up and the relevant report of plant regeneration.
Marine alga Study on tissue culture is from the sixties in last century, be in progress rapider after the eighties, Zhao Huandeng and Zhang Xuecheng (1981) polishing, Lu Chengqing (nineteen eighty-three) method of rotting, it is unicellular and all regenerate strain that Saga (1984) is separated to porphyra yezoensis with enzyme process.Country's " the Seventh Five-Year Plan " brainstorm subject " laver cell nursery " born by the Institute of Oceanology of the Chinese Academy of Sciences, Shanghai Aquatic Products Univ. 9CN) and Qingdao Marine University passed through to check and accept in nineteen ninety.Expert thinks, this problem utilizes cell culture technology to carry out nursery, greatly shortens seedling raise period than conventional method, and seedling rearing room floor space is little, and cost is low, has certain Social and economic benef@, reaches the international leading level.Marine alga tissue cultures develops more ripe in laver, and research in the China such as sea-tangle and undaria pinnitafida Main Economic marine alga is deep not enough.
Current kelp germplasm is preserved and breeding, raising seedling mainly contains two kinds of approach: a kind of is adopt ripe sporophyte to carry out conservation, fine-variety breeding and nursery production by the mode of generation seed selection, nursery, namely this conservation and Seedling production mode inevitably cause loss and the degeneration of breeding merit in the process selected by generation, carry out breeding of new variety by the method and then have that breeding cycle is long, Breeding Efficiency is low, the drawback that labour intensity is large.Another kind of approach is based on gametophytic conservation and breeding, raising seedling mode.This mode diffuses trip spore by ripe sporophyte, in time developing into the male and female gametophytes can offered an explanation under the microscope, its single separation is preserved, is cultivated, can be used for the long-term preservation of kind of matter, breeding and nursery and produce.Compared with the former, this mode has that varietal purity is high, long service life, nursery is by season and the restriction of sea area condition and the advantage such as breeding cycle is shorter.Above two main laminaria conservations, breeding and nursery approach all have premised on sporangial ripe sporophyte by long, and some phenotypic characters obtained by unstable mode (as radiation, mutagenesis or spontaneous mutation etc.) or alternate manner are given prominence to but long sporangial individuality, there is no effective preserving seed and offspring seed cultivation method at present.
Summary of the invention
The technical problem to be solved in the present invention is: provide a kind of method utilizing sea-tangle explant callus to carry out preserving seed and larval rearing thereof.
Technical solution of the present invention is: the method that the first kelp germplasm is preserved, and the steps include:
(1) pretreatment of sea-tangle children spore explant:
A, sea-tangle juvenile sporophyte, after sterilizing seawater flushing, obtain sea-tangle children spore stripping and slicing with sterilizing blade the holdfast of sea-tangle juvenile sporophyte and shank excision, then remove sea-tangle children spore diced facets foreign material, and with the young spore stripping and slicing of sterilizing seawater flushing sea-tangle,
B, sea-tangle children spore stripping and slicing to be moved in superclean bench, first soak at twice with the liquor kalii iodide that concentration is 1.5%, each 7 ~ 10 minutes sterilizings, move into again in dual anti-solution, also soak at twice, each 7 ~ 10 minutes sterilizings, then sterilizing seawater flushing is used 2 ~ 3 times
C, with sterilizing blade by sea-tangle children spore stripping and slicing by base portion, 1/4 ~ 1/5 of whole sea-tangle children spore stripping and slicing is cut, obtain sea-tangle children spore growth portion stripping and slicing, use sterilizing cutter by sea-tangle children spore growth portion stripping and slicing edge trimming again, make sea-tangle children spore growth portion stripping and slicing outward flange expose new section, it is 2 ~ 3mm that area is cut in the sea-tangle children spore growth portion stripping and slicing of outward flange being exposed new section again
2bulk obtain sea-tangle children spore explant;
(2) induction of callus: sea-tangle children spore explant is placed in culture dish, add the PESI culture fluid that concentration is 2%, then cultivate in illumination box, cultivation temperature is 15 DEG C, intensity of illumination is 1500 ~ 2000 lux, and the photoperiod is 10L:14D, and described PESI culture fluid is changed once for every 7 days, sea-tangle children spore explant was cultivated through 1 ~ 2 month, obtained sea-tangle callus;
(3) preservation of callus: the sea-tangle callus sterilizing blade of turning out is cut by sea-tangle children spore explant and collected, proceed to 100ml conical flask to cultivate in illumination box, medium to be concentration be 2% PESI culture fluid, cultivation temperature is 5 ~ 10 DEG C, intensity of illumination is 500 ~ 1000 lux, photoperiod is 10L:14D, and described PESI culture fluid is changed once for every 30 days, and the sea-tangle callus obtained with this understanding can be preserved for a long time as kelp germplasm material.
The method that the second kelp germplasm of the present invention is preserved, the steps include:
(1) pretreatment of sea-tangle megaspore explant:
Sea-tangle macrosporinite growing part, after cleaning, is cut and is obtained the stripping and slicing of sea-tangle macrosporinite growing part by a, sea-tangle macrosporinite, removes the foreign material of sea-tangle macrosporinite growing part diced facets, and uses sterilizing seawater flushing,
B, the stripping and slicing of sea-tangle macrosporinite growing part to be moved in superclean bench, first soak at twice with the liquor kalii iodide that concentration is 1.5%, each 7 ~ 10 minutes sterilizings, move in dual anti-solution again, also soak at twice, each 7 ~ 10 minutes sterilizings, then use sterilizing seawater flushing 2 ~ 3 times
C, by sea-tangle macrosporinite growing part stripping and slicing edge trimming, make sea-tangle macrosporinite growing part stripping and slicing outward flange expose new section, it is 2 ~ 3mm that area is cut in the sea-tangle macrosporinite growing part stripping and slicing of outward flange being exposed new section again
2bulk obtain sea-tangle megaspore explant;
(2) induction of callus: sea-tangle megaspore explant is placed in culture dish, add macrosporinite culture fluid, macrosporinite culture fluid is the mixed liquor containing PESI and NAA, the concentration of the PESI in mixed liquor is 2%, NAA final concentration is 1 ~ 1.86mg/L, then cultivate in illumination box, cultivation temperature is 15 DEG C, intensity of illumination is 1500 ~ 2000 lux, photoperiod is 10L:14D, mixed liquor is changed once for every 7 days, and sea-tangle megaspore explant was cultivated through 1 ~ 2 month, obtained sea-tangle callus;
(3) preservation of callus: by the sea-tangle callus sterilizing blade of turning out by sea-tangle megaspore explant cutting and collecting, proceed to 100ml conical flask to cultivate in illumination box, medium to be concentration be 2% PESI culture fluid, cultivation temperature is 5 ~ 10 DEG C, intensity of illumination is 500 ~ 1000 lux, photoperiod is that 10L:14D, PESI culture fluid is changed once for every 30 days, and the sea-tangle callus obtained with this understanding can be preserved for a long time as kelp germplasm material.
The Compound mixed solution method of PESI and NAA of above-mentioned steps (2): take 10mg methyl α-naphthyl acetate, first dissolve with the sodium hydroxide solution that 500 μ l concentration are 1mol/l, add distilled water again and be settled to 10ml, then obtain with the filter membrane suction filtration sterilizing of 0.22 μm the naphthalene acid solution that concentration is 1mg/ml; Described naphthalene acid solution being added to concentration is in the PESI culture fluid of 2% again, and every 100ml concentration is methyl α-naphthyl acetate 100 ~ 186 μ l adding concentration 1mg/ml in the PESI culture fluid of 2%.
Above-mentioned concentration is the collocation method of the PESI culture fluid of 2%: take 350mg sodium nitrate, the sodium glycero-phosphate of 50mg, the iron edta sodium salt of 2.5mg, the trishydroxymethylaminomethane of 500mg, the potassium iodide of 100 μ g, P-II molten metal measuring 25ml adds described sodium nitrate, sodium glycero-phosphate, iron edta sodium salt, trishydroxymethylaminomethane, after potassium iodide is dissolved, 100ml is settled to distilled water, adjusted to ph is the 7.8 PESI culture fluids namely obtaining that concentration is 2%, the collocation method of described P-II molten metal is: the disodium ethylene diamine tetraacetate taking 100mg, the iron chloride of 1mg, the boric acid of 20mg, the manganese chloride of 4mg, the zinc chloride of 0.5mg, the cobalt chloride of 0.1mg, dissolve with distilled water, and be settled to 100ml with distilled water.
Above-mentioned concentration is the collocation method of the liquor kalii iodide of 1.5%: take 1.5g potassium iodide, after dissolving, and is settled to 100ml with sterilizing seawater and namely obtains the liquor kalii iodide that concentration is 1.5% with sterilizing seawater; The collocation method of described dual anti-solution: take 0.08g Ciprofloxacin Lactate, 0.1g penicillin, dissolves with distilled water, and is settled to 1000ml with distilled water and namely obtains dual anti-solution.
It is 103.4kPa that above-mentioned sterilizing blade adopts at pressure, and temperature is sterilizing 30 minutes under the condition of 121 DEG C.
Kelp seedling breeding method of the present invention, the steps include:
(1) the spreading cultivation of callus: the sea-tangle callus of above-mentioned acquisition is shredded, proceeding to concentration is in the PESI culture fluid of 2%, cultivation temperature is 15 DEG C, intensity of illumination is 2000 ~ 3000lux, photoperiod is 10L:14D, described PESI culture fluid is changed once for every 7 days, and through the cultivation of 20 ~ 30 days, sea-tangle callus was grown up; Again collect the sea-tangle callus after growing up and shred, then to proceed to concentration be again cultivate in the PESI culture fluid of 2%, through the cultivation of 20 ~ 30 days, sea-tangle callus was grown up again; Repeat above operation continuously, sea-tangle callus can keep vigorous growth, obtains the callus after spreading cultivation;
(2) seedling differentiation of callus: collect the callus after spreading cultivation and smash, change concentration be 2% PESI culture fluid carry out renewal cultivation, the temperature of renewal cultivation is 15 DEG C, intensity of illumination is 2000 ~ 3000lux, photoperiod is 10L:14D, callus renewal cultivation is after one week, proceed in low temperature nursery storehouse, be inoculated on breeding screen, with fluorescent lamp or natural daylight for light source, initial intensity of illumination is 2000 ~ 2500lux, water temperature is 8 ~ 10 DEG C, culture fluid is replaced by N/P nutrient solution, quiescent culture has a large amount of juvenile sporophyte to occur for 7 ~ 10 days afterwards as seen, now can start to change water or flowing water, later stage condition of culture is produced with conventional seedbed system, laminaria production seedling can be obtained through the cultivation of 1 ~ 2 month.
Prepare the method for above-mentioned N/P nutrient solution: take 121 grams of sodium nitrate, after dissolving with sterilizing seawater, then continuation sterilizing seawater is settled to 500 ml and obtains sodium nitrate solution; Take 17.5 grams of potassium dihydrogen phosphates, after dissolving with sterilizing seawater, then be settled to 1000 ml with sterilizing seawater and obtain potassium dihydrogen phosphate; Measure potassium dihydrogen phosphate described in sodium nitrate solution described in 1 ml and 1 ml respectively, be added in the sterilizing seawater of 4000 ml, shake up and can obtain described N/P nutrient solution.
Technique effect of the present invention is: the present invention's liquid nutrient medium induction kelp spore explant (sea-tangle children's spore explant and sea-tangle megaspore explant) can obtain the high efficiency induction of sea-tangle callus; Give certain condition of culture, namely callus can be used as germplasm materials and preserves for a long time, and carried out spreading cultivation and breaking up by callus, can cultivate a large amount of kelp seedlings, these kelp seedlings can be used for breeding production.
Sea-tangle explant callus preserving seed of the present invention and larval rearing thereof, not by season and climatic influences, just can reach plant corpus fast breeding object in the short time, and can produce kind of the yielding ability sea-tangle seedling that property is pure, quality better, output are high.Research shows, is in the plant living cells of ex vivo situation, under the effect of suitable nutriment, hormone and other external conditions, just may show totipotency, develop into complete plant.The present invention does not limit by ripe sporophyte, the sporophyte growing part of each growth period all can be used as explant induction and goes out callus, the callus obtained not only can be used as germplasm materials and preserves for a long time, and can spread cultivation further and break up, and produces for breeding and nursery.The present invention have applicability strong, be easy to Artificial Control, draw materials less, efficiency is high, simple to operate, be convenient to the feature promoted, for kelp germplasm preservation and breeding, raising seedling open up a new way, simultaneously also can be cytology and genetics research provides material, in the research carrying out secondary substance synthesis and specific drugs conversion utilizing sea-tangle tissue and cell cultivation, also there is potential using value.
Embodiment
Be described in detail below in conjunction with specific embodiment:
Embodiment 1
Embodiment 1 utilizes sea-tangle juvenile sporophyte (kelp seedling) to carry out pretreatment, the induction of callus and preservation, then spread cultivation and seedling differentiation by callus.
(1) pretreatment of sea-tangle children spore explant: select the complete hurtless measure of blade, health and lovely luster, sea-tangle juvenile sporophyte that algae pollution of mixing is few, length is between 1.0 ~ 2.5cm.Sea-tangle juvenile sporophyte, after sterilizing seawater repeatedly rinses, excises sea-tangle juvenile sporophyte holdfast and shank with sterilizing blade; With cotton swab gently wiping sea-tangle juvenile sporophyte blade surface to remove foreign material, and use sterilizing seawater flushing, can repeat wiping several all over for subsequent use after rinsing, when noting wiping, dynamics wants light, damages sea-tangle juvenile sporophyte blade again.
Operator's both hands are the alcohol disinfecting of 75% by concentration in advance, then sea-tangle juvenile sporophyte blade good for wiping is moved in the superclean bench of prior ultraviolet disinfection, it is first liquor kalii iodide (KI) the total immersion bubble 15min sterilizing at twice of 1.5% by concentration, move in dual anti-solution again, also soak at twice, coprocessing 15min, then uses sterilizing seawater flushing 2 ~ 3 times, is rinsed out by residual dual anti-solution.With sterilizing blade by sea-tangle juvenile sporophyte blade by base portion, 1/4 ~ 1/5 of whole blade cuts and obtains the stripping and slicing of sea-tangle juvenile sporophyte growing part, namely cuts the growing part that material is sea-tangle juvenile sporophyte.The present embodiment sea-tangle juvenile sporophyte liquor kalii iodide soaks at twice, each 7 ~ 10 minutes sterilizings, then is moved in dual anti-solution by sea-tangle juvenile sporophyte, also soaks at twice, each 7 ~ 10 minutes.
Get the stripping and slicing of sea-tangle juvenile sporophyte growing part, with sterilizing cutter, sea-tangle juvenile sporophyte growing part stripping and slicing four edges cut away a bit all again, expose new section, after the sea-tangle juvenile sporophyte growing part stripping and slicing of reservation is cut into 2 ~ 3mm again
2bulk do the material of tissue cultures as sea-tangle children spore explant.
(2) induction of callus: cultivating sea-tangle children spore explant at diameter is in the glass culture dish of 9cm, adds the PESI culture fluid 30ml that concentration is 2% in culture dish in advance, then with tweezers, 7 ~ 8 pieces of sea-tangle children spore explants are put into culture fluid; The glass culture dish being placed with PESI culture fluid and sea-tangle children spore explant is cultivated in illumination box, cultivation temperature is 15 DEG C, intensity of illumination is 1500 ~ 2000lux, photoperiod is 10L:14D(illumination 10 hours, dark 14 hours, circulation conversion), PESI culture fluid is changed once for every 7 days, each full dose is changed, and carries out structure observation.Sea-tangle children spore explant was cultivated through 1 ~ 2 month, can obtain sea-tangle callus.
(3) preservation of callus: the sea-tangle callus sterilizing blade of turning out is cut by sea-tangle children spore explant and collected, proceed to 100ml conical flask to cultivate in illumination box, medium to be concentration be 2% PESI culture fluid, cultivation temperature is 5 ~ 10 DEG C, intensity of illumination is 500 ~ 1000lux, photoperiod is that 10L:14D, PESI culture fluid is changed once for every 30 days, and each full dose is changed.With this understanding, sea-tangle callus growth is comparatively slow, can preserve for a long time as germplasm materials.
(4) the spreading cultivation of callus: the chopping of sea-tangle callus scalpel or high-speed tissue mashing machine are smashed, proceeding to fresh concentration is in the PESI culture fluid of 2%, cultivate in illumination box, cultivation temperature is 15 DEG C, intensity of illumination is 2000 ~ 3000lux, photoperiod is 10L:14D, and culture fluid is changed once for 7 days, and each full dose is changed.Through the cultivation of about 15 ~ 20 days, sea-tangle callus is grown up, again can collect sea-tangle callus and smash with scalpel chopping or high-speed tissue mashing machine again, proceeding to fresh concentration is again in the PESI culture fluid of 2%, cultivate in illumination box, cultivation temperature is 15 DEG C, intensity of illumination is 2000 ~ 3000lux, photoperiod is 10L:14D, and culture fluid 7 days is changed once, then through the cultivation of 15 ~ 20 days, repeat above operation again, such sea-tangle callus can keep vigorous growth, can reach the object expanding and cultivate, use in order to follow-up nursery.
(5) seedling differentiation of callus
When sea-tangle callus spreads cultivation to requirement, collected and fully smashed with high-speed tissue mashing machine, and the concentration more renewed is the PESI culture fluid of 2%, renewal cultivation is after one week, proceed in low temperature nursery storehouse, be inoculated on breeding screen, with fluorescent lamp or natural daylight for light source, initial intensity of illumination is 2000 ~ 2500lux, water temperature is 8 ~ 10 DEG C, culture fluid is replaced by N/P nutrient solution, quiescent culture has a large amount of juvenile sporophyte to occur for 7 ~ 10 days afterwards as seen, at this moment can start regularly replace new culture fluid or carry out flowing water cultivation, later stage condition of culture is produced with conventional kelp seedling cultivation, the cultivation water temperature that conventional kelp seedling cultivation is produced: 7 ~ 8 DEG C.Intensity of illumination: take natural daylight as light source, manual adjustment intensity of illumination, when sea-tangle juvenile sporophyte length is less than 0.2mm, the highest light intensity 3500 lux, average intensity 1700 ~ 1800 lux; Sea-tangle juvenile sporophyte length when 0.2 ~ 0.3mm, the highest light intensity 4000 lux, average intensity 1800 ~ 2000 lux; Sea-tangle juvenile sporophyte length when 0.3 ~ 3mm, the highest light intensity 4000 lux, average intensity 2000 ~ 2200 lux; Sea-tangle juvenile sporophyte length when 3 ~ 5 mm, the highest light intensity 5000 lux, average intensity 2200 ~ 2500 lux; Sea-tangle juvenile sporophyte length when 5 ~ 10mm, the highest light intensity 5500 lux, average intensity 2500 ~ 2800 lux; Sea-tangle juvenile sporophyte length is the highest light intensity 6000 more than lux when more than 1cm, average intensity 3000 more than lux.Day quantity of exchanged water: before sea-tangle juvenile sporophyte length reaches 5mm, every day, quantity of exchanged water was 50% of total water body; After sea-tangle juvenile sporophyte length reaches 5mm, every day, quantity of exchanged water was 75% of total water body; After sea-tangle juvenile sporophyte length reaches 10mm, every day, quantity of exchanged water was 100% of total water body.Clear pond: for avoiding assorted algae and bacterium amount reproduction, thoroughly scrubs according to water quality condition and juvenile sporophyte growing state and cultivates pond once for every about 10 days.Flow velocity and flow time: when flowing water is cultivated, front 24 ~ 48 hr flow rate are less than 0.05m/s, and after 72 hours, flow velocity is less than 0.1m/s.Seedling curtain is done the wash: sea-tangle juvenile sporophyte length did not scrub seedling curtain before 0.2mm, and sea-tangle juvenile sporophyte length scrubs initial pressure 0.5KG when 0.2 ~ 0.3mm, scrubs pressure and is increased to 1.0KG gradually; Sea-tangle juvenile sporophyte length is when 0.3 ~ 3mm, and scrubbing pressure is 1.5 KG; Sea-tangle juvenile sporophyte length is when 3 ~ 5 mm, and scrubbing pressure is 2.0 KG; Sea-tangle juvenile sporophyte length, at 5 more than mm, scrubs that pressure is maximum is no more than 2.5 KG.The production kelp seedling of length 1 ~ 2cm can be obtained through the cultivation of 1 ~ 2 month.
Embodiment 2
Embodiment 2 utilizes sea-tangle macrosporinite (adult sea-tangle) to carry out pretreatment, the induction of callus and preservation, then spread cultivation and seedling differentiation by callus.
(1) pretreatment of explant: select the complete hurtless measure of blade, health and lovely luster, sea-tangle macrosporinite (adult sea-tangle) that assorted algae pollution is few be material.After sea-tangle is at sea tentatively cleaned with seawater by sea-tangle macrosporinite, take use for laboratory scissors sea-tangle macrosporinite growing part is cut, with absorbent cotton wiping sea-tangle macrosporinite growing part repeatedly, to remove the foreign material on sea-tangle macrosporinite growing part surface, and repeatedly rinse rear for subsequent use with sterilizing seawater.
Operator's both hands concentration is 75% alcohol disinfecting, sea-tangle macrosporinite growing part good for wiping is moved in the superclean bench of prior ultraviolet disinfection, first with concentration be 1.5% potassium iodide (KI) solution at twice total immersion steep 15 minutes sterilizings, move in dual anti-solution again, also soak at twice, coprocessing 15 minutes, with sterilizing seawater flushing 2 ~ 3 times, rinses out residual dual anti-solution.
Sea-tangle macrosporinite growing part four edges are cut away a bit with scalpel, expose new section by sea-tangle macrosporinite growing part stripping and slicing all again, after area is cut in the stripping and slicing of exposing new section is again 2 ~ 3mm
2block sea-tangle megaspore explant do the material of tissue cultures.
(2) induction of callus: cultivating sea-tangle megaspore explant at diameter is in the glass culture dish of 9cm, sea-tangle megaspore explant culture fluid is containing PESI and NAA(methyl α-naphthyl acetate) mixed liquor, the concentration of the PESI in mixed liquor is 2%, NAA final concentration is 1 ~ 1.86mg/L; Each glass culture dish adds PESI and NAA mixed liquor and is about 30ml, and then puts into 7 ~ 8 pieces of sea-tangle megaspore explants; Cultivate in illumination box, cultivation temperature is 15 DEG C, and intensity of illumination is 1500 ~ 2000lux, and the photoperiod is 10L:14D, and culture fluid is changed once for every 7 days, and each full dose is changed, and carries out structure observation.Sea-tangle megaspore explant was cultivated through 1 ~ 2 month, can obtain sea-tangle callus.
(3) preservation of callus: by the sea-tangle callus sterilizing blade of turning out by sea-tangle megaspore explant cutting and collecting, proceed to 100ml conical flask to cultivate in illumination box, medium to be concentration be 2% PESI culture fluid, cultivation temperature is 5 ~ 10 DEG C, intensity of illumination is 500 ~ 1000lux, photoperiod is 10L:14D, and culture fluid is changed once for every 30 days, and each full dose is changed.With this understanding, sea-tangle callus growth is comparatively slow, can preserve for a long time as germplasm materials.
(4) the spreading cultivation of callus: by the chopping of sea-tangle callus scalpel or smash with high-speed tissue mashing machine, proceeding to fresh concentration is in the PESI culture fluid of 2%, cultivation temperature is 15 DEG C, intensity of illumination is 2000 ~ 3000lux, photoperiod is 10L:14D, culture fluid is changed once for every 7 days, and each full dose is changed.Through the cultivation of about 20 ~ 30 days, callus is grown up, again can collect and smash with scalpel chopping or high-speed tissue mashing machine, proceeding to fresh concentration is cultivate in the PESI culture fluid of 2%, again through the cultivation of 20 ~ 30 days, then repeat above operation, such sea-tangle callus can keep vigorous growth, the object expanding and cultivate can be reached, use in order to follow-up nursery.
(5) seedling differentiation of callus
When sea-tangle callus spread cultivation to requirement (spread cultivation to can nursery produce amount, generally The more the better, cannot be used up and can continue long-term preservation) after, sea-tangle callus after spreading cultivation is collected and fully smashes with high-speed tissue mashing machine, and the concentration more renewed is the PESI culture fluid of 2%, renewal cultivation is after one week, proceed in low temperature nursery storehouse, be inoculated on breeding screen, with fluorescent lamp or natural daylight for light source, initial intensity of illumination is 2000 ~ 2500lux, water temperature is 8 ~ 10 DEG C, culture fluid is replaced by N/P nutrient solution, quiescent culture has a large amount of sea-tangle juvenile sporophyte to occur for 7 ~ 10 days afterwards as seen, now can start to change water or flowing water, later stage condition of culture is produced with conventional seedbed system.The production kelp seedling of length 1 ~ 2 centimetre can be obtained through the cultivation of 1 ~ 2 month.
Apparatus, the auxiliary materials such as embodiment cotton swab used, absorbent cotton, glass culture dish, sterilizing blade, tweezers, scalpel and high-speed tissue mashing machine all shift to an earlier date autoclaving with vertical pressure steam sterilization pan, sterilization pressure is 103.4kPa, sterilising temp is 121 DEG C, sterilization time 30 minutes.
The embodiment of the present invention adopts following experimental instrument:
A, illumination box: MGC-300A type, the permanent Science and Technology Ltd. in Shanghai one
B, microscope: CX22 type, OLYMPUS
C, refrigerator: BCD-216TX type, Qingdao HaiEr Co., Ltd
D, high-speed tissue mashing machine: DS-1 type, Shanghai Sample Model Factory
E, superclean bench: SW-CJ-ID type, Purifying Equipment Co., Ltd., Suzhou
F, vertical pressure steam sterilization pan: YXQ-LS-75SII, Medical Equipment Plant of Shanghai Boxun Industrial Co., Ltd.
G, pH meter: PB-10, Sai Duolisi scientific instrument (Beijing) Co., Ltd
The embodiment of the present invention adopts following experiment reagent
1) sterilizing seawater seawater of making a living boils 2 minutes, and naturally cools to room temperature.
2) concentration is the liquor kalii iodide of 1.5%: take 1.5g potassium iodide (molecular formula KI, Shanghai Chemical Reagent Co., Ltd., Sinopharm Group produces, and analyzes pure), dissolve and be settled to 100ml with sterilizing seawater.
3) dual anti-solution: (molecular formula is C to take 0.08g Ciprofloxacin Lactate soluble powder
17h
18fN
3o
3, Guangdong Ke Run Biology Pharmacy Co., Ltd produces), (molecular formula is C to 0.1g penicillin
16h
17n
2naO
4s, Lukang Medical Co., Ltd., Shandong produces, 800,000 units), dissolve with distilled water and be settled to 1000ml.
4) concentration is the PESI culture fluid of 2%: (molecular formula is NaNO to take 350mg sodium nitrate
3, Shanghai Chemical Reagent Co., Ltd., Sinopharm Group produces, and analyzes pure), (molecular formula is Na to 50mg sodium glycero-phosphate
2c
3h
5(OH)
2pO
45 H
2o, Chengdu section dragon chemical reagent factory is produced, and analyzes pure), (molecular formula is C to 2.5mg iron edta sodium salt
10h
12feN
2na0, MDBio, Inc. produce, and analyze pure), (molecular formula is C to 500mg trishydroxymethylaminomethane
4h
11nO
3abbreviation is Tris, MDBio, Inc. produce, and analyze pure), (molecular formula is KI to 100 μ g potassium iodide, Shanghai Chemical Reagent Co., Ltd., Sinopharm Group produces, and analyzes pure), after P-II molten metal measuring 25ml adds dissolving, be settled to 100ml with distilled water, and be 7.8 by the sodium hydroxide solution adjusted to ph that concentration is 1 mol/l.When by concentration being the sodium hydroxide solution adjust ph of 1 mol/l, while add a sodium hydroxide solution limit pH meter to survey pH, until solution adjusted to ph is 7.8.
5) P-II molten metal: (molecular formula is C to take 100mg disodium ethylene diamine tetraacetate
10h
14n
2o
8na
22H
2o, Shanghai Chemical Reagent Co., Ltd., Sinopharm Group produces, and analyzes pure), (molecular formula is FeCl to 1mg iron chloride
3, Shanghai Chemical Reagent Co., Ltd., Sinopharm Group produces, and analyzes pure), 20mg boric acid (molecular formula is H BO, and Shanghai Chemical Reagent Co., Ltd., Sinopharm Group produces, and analyzes pure), (molecular formula is MnCl to 4mg manganese chloride
2, Shanghai Chemical Reagent Co., Ltd., Sinopharm Group produces, and analyzes pure), (molecular formula is ZnCl to 0.5mg zinc chloride
2, Shanghai Chemical Reagent Co., Ltd., Sinopharm Group produces, and analyzes pure), (molecular formula is CoCl to 0.1mg cobalt chloride
2, Shanghai Chemical Reagent Co., Ltd., Sinopharm Group produces, and analyzes pure), dissolve with distilled water and be settled to 100ml.
6) compound concentration is the methyl α-naphthyl acetate (NAA) of 1mg/ml: take 10mg methyl α-naphthyl acetate powder (molecular formula abbreviation NAA, Beijing Suo Laibao Science and Technology Ltd. produces), first dissolve with the sodium hydroxide solution that 500 μ l concentration are 1mol/l, add distilled water again and be settled to 10ml, with the filter membrane suction filtration sterilizing of 0.22 μm.This solution is used for adding in 2%PESI culture fluid, and every 100ml concentration is methyl α-naphthyl acetate 100 ~ 186 μ l(microlitre adding concentration 1mg/ml in the PESI culture fluid of 2%).
7) compound concentration is the sodium hydroxide solution of 1mol/l: take 4g sodium hydroxide powder (molecular formula NaOH, Shanghai Chemical Reagent Co., Ltd., Sinopharm Group produces, analyze pure), dissolve with distilled water and be settled to 100ml, for preparing the methyl α-naphthyl acetate of 1mg/ml.
8) sodium nitrate solution is prepared: take 121 grams of sodium nitrate (molecular formula NaNO
3, Shanghai Chemical Reagent Co., Ltd., Sinopharm Group produces, and analyzes pure), dissolve with sterilizing seawater and be settled to 500 ml, for preparation containing N/P culture fluid.
9) potassium dihydrogen phosphate is prepared: take 17.5 grams of potassium dihydrogen phosphate (molecular formula KH
2pO
4, Shanghai Chemical Reagent Co., Ltd., Sinopharm Group produces, and analyzes pure), dissolve with sterilizing seawater and be settled to 1000 ml, for preparing N/P culture fluid.
10) preparation is containing N/P culture fluid (nitrogen phosphorus culture fluid): measure above-mentioned 1 ml sodium nitrate solution and 1 ml potassium dihydrogen phosphate respectively, be added to 4000 ml through boiling 2 minutes and naturally cooling in the sterilizing seawater of room temperature, shake up for subsequent use.r b
In Plant Tissue Breeding, the various organs of use, tissue and cell are referred to as explant.Callus refers to that explant is because of injured or when cultured in vitro, and its undifferentiated cell and the cell broken up carry out the division growth that enlivens and a kind of tissue without ad hoc structure and function formed.Callus cell can carry out preserving, cultivate and increasing, and cell has totipotency through dedifferentiation simultaneously, can under proper culture conditions through being differentiated to form new plant corpus again.Like this through the induction of callus, the amplification of cell, then break up, just can reach the object of plant corpus fast asexual propagation.
Claims (8)
1. a method for kelp germplasm preservation, the steps include:
(1) pretreatment of sea-tangle children spore explant:
A, sea-tangle juvenile sporophyte, after sterilizing seawater flushing, obtain sea-tangle children spore stripping and slicing with sterilizing blade the holdfast of sea-tangle juvenile sporophyte and shank excision, then remove sea-tangle children spore diced facets foreign material, and with the young spore stripping and slicing of sterilizing seawater flushing sea-tangle,
B, sea-tangle children spore stripping and slicing to be moved in superclean bench, first soak at twice with the liquor kalii iodide that concentration is 1.5%, each 7 ~ 10 minutes sterilizings, move into again in dual anti-solution, also soak at twice, each 7 ~ 10 minutes sterilizings, then sterilizing seawater flushing is used 2 ~ 3 times
C, with sterilizing blade by sea-tangle children spore stripping and slicing by base portion, 1/4 ~ 1/5 of whole sea-tangle children spore stripping and slicing is cut, obtain sea-tangle children spore growth portion stripping and slicing, use sterilizing cutter by sea-tangle children spore growth portion stripping and slicing edge trimming again, make sea-tangle children spore growth portion stripping and slicing outward flange expose new section, it is 2 ~ 3mm that area is cut in the sea-tangle children spore growth portion stripping and slicing of outward flange being exposed new section again
2bulk obtain sea-tangle children spore explant;
(2) induction of callus: sea-tangle children spore explant is placed in culture dish, add the PESI culture fluid that concentration is 2%, then cultivate in illumination box, cultivation temperature is 15 DEG C, intensity of illumination is 1500 ~ 2000lux, and the photoperiod is 10L:14D, and described PESI culture fluid is changed once for every 7 days, sea-tangle children spore explant was cultivated through 1 ~ 2 month, obtained sea-tangle callus;
(3) preservation of callus: the sea-tangle callus sterilizing blade of turning out is cut by sea-tangle children spore explant and collected, proceed to 100ml conical flask to cultivate in illumination box, medium to be concentration be 2% PESI culture fluid, cultivation temperature is 5 ~ 10 DEG C, intensity of illumination is 500 ~ 1000lux, photoperiod is 10L:14D, and described PESI culture fluid is changed once for every 30 days, and the sea-tangle callus obtained with this understanding can be preserved for a long time as kelp germplasm material.
2. a method for kelp germplasm preservation, the steps include:
(1) pretreatment of sea-tangle megaspore explant:
Sea-tangle macrosporinite growing part, after cleaning, is cut and is obtained the stripping and slicing of sea-tangle macrosporinite growing part by a, sea-tangle macrosporinite, removes the foreign material of sea-tangle macrosporinite growing part diced facets, and uses sterilizing seawater flushing,
B, the stripping and slicing of sea-tangle macrosporinite growing part to be moved in superclean bench, first soak at twice with the liquor kalii iodide that concentration is 1.5%, each 7 ~ 10 minutes sterilizings, move in dual anti-solution again, also soak at twice, each 7 ~ 10 minutes sterilizings, then use sterilizing seawater flushing 2 ~ 3 times
C, by sea-tangle macrosporinite growing part stripping and slicing edge trimming, make sea-tangle macrosporinite growing part stripping and slicing outward flange expose new section, it is 2 ~ 3mm that area is cut in the sea-tangle macrosporinite growing part stripping and slicing of outward flange being exposed new section again
2bulk obtain sea-tangle megaspore explant;
(2) induction of callus: sea-tangle megaspore explant is placed in culture dish, add macrosporinite culture fluid, macrosporinite culture fluid is the mixed liquor containing PESI and NAA, the concentration of the PESI in mixed liquor is 2%, NAA final concentration is 1 ~ 1.86mg/L, then cultivate in illumination box, cultivation temperature is 15 DEG C, intensity of illumination is 1500 ~ 2000lux, photoperiod is 10L:14D, mixed liquor is changed once for every 7 days, and sea-tangle megaspore explant was cultivated through 1 ~ 2 month, obtained sea-tangle callus;
(3) preservation of callus: by the sea-tangle callus sterilizing blade of turning out by sea-tangle megaspore explant cutting and collecting, proceed to 100ml conical flask to cultivate in illumination box, medium to be concentration be 2% PESI culture fluid, cultivation temperature is 5 ~ 10 DEG C, intensity of illumination is 500 ~ 1000lux, photoperiod is that 10L:14D, PESI culture fluid is changed once for every 30 days, and the sea-tangle callus obtained with this understanding can be preserved for a long time as kelp germplasm material.
3. the method for a kind of kelp germplasm preservation according to claim 2, it is characterized in that: the Compound mixed solution method of PESI and NAA of described step (2): take 10mg methyl α-naphthyl acetate, first dissolve with the sodium hydroxide solution that 500 μ l concentration are 1mol/l, add distilled water again and be settled to 10ml, then obtain with the filter membrane suction filtration sterilizing of 0.22 μm the naphthalene acid solution that concentration is 1mg/ml; Described naphthalene acid solution being added to concentration is in the PESI culture fluid of 2% again, and every 100ml concentration is methyl α-naphthyl acetate 100 ~ 186 μ l adding concentration 1mg/ml in the PESI culture fluid of 2%.
4. a kind of method that kelp germplasm is preserved according to claim 1 or 2, it is characterized in that: to be the collocation method of the PESI culture fluid of 2% be described concentration: take 350mg sodium nitrate, the sodium glycero-phosphate of 50mg, the iron edta sodium salt of 2.5mg, the trishydroxymethylaminomethane of 500mg, the potassium iodide of 100 μ g, P-II molten metal measuring 25ml adds described sodium nitrate, sodium glycero-phosphate, iron edta sodium salt, trishydroxymethylaminomethane, after potassium iodide is dissolved, 100ml is settled to distilled water, adjusted to ph is the 7.8 PESI culture fluids namely obtaining that concentration is 2%, the collocation method of described P-II molten metal is: the disodium ethylene diamine tetraacetate taking 100mg, the iron chloride of 1mg, the boric acid of 20mg, the manganese chloride of 4mg, the zinc chloride of 0.5mg, the cobalt chloride of 0.1mg, dissolve with distilled water, and be settled to 100ml with distilled water.
5. a kind of method that kelp germplasm is preserved according to claim 1 or 2, it is characterized in that: described concentration is the collocation method of the liquor kalii iodide of 1.5%: take 1.5g potassium iodide, after dissolving with sterilizing seawater, and be settled to 100ml with sterilizing seawater and namely obtain the liquor kalii iodide that concentration is 1.5%; The collocation method of described dual anti-solution: take 0.08g Ciprofloxacin Lactate, 0.1g penicillin, dissolves with distilled water, and is settled to 1000ml with distilled water and namely obtains dual anti-solution.
6. a kind of method that kelp germplasm is preserved according to claim 1 or 2, is characterized in that: it is 103.4kPa that described sterilizing blade adopts at pressure, and temperature is sterilizing 30 minutes under the condition of 121 DEG C.
7. a kelp seedling breeding method, the steps include:
(1) the spreading cultivation of callus: the sea-tangle callus chopping that claim 1 or 2 is obtained, proceeding to concentration is in the PESI culture fluid of 2%, cultivation temperature is 15 DEG C, intensity of illumination is 2000 ~ 3000lux, photoperiod is 10L:14D, described PESI culture fluid is changed once for every 7 days, and through the cultivation of 20 ~ 30 days, sea-tangle callus was grown up; Again collect the sea-tangle callus after growing up and shred, then to proceed to concentration be again cultivate in the PESI culture fluid of 2%, through the cultivation of 20 ~ 30 days, sea-tangle callus was grown up again; Repeat above operation continuously, sea-tangle callus can keep vigorous growth, obtains the callus after spreading cultivation;
(2) seedling differentiation of callus: collect the callus after spreading cultivation and smash, change concentration be 2% PESI culture fluid carry out renewal cultivation, the temperature of renewal cultivation is 15 DEG C, intensity of illumination is 2000 ~ 3000lux, photoperiod is 10L:14D, callus renewal cultivation is after one week, proceed in low temperature nursery storehouse, be inoculated on breeding screen, with fluorescent lamp or natural daylight for light source, initial intensity of illumination is 2000 ~ 2500lux, water temperature is 8 ~ 10 DEG C, culture fluid is replaced by N/P nutrient solution, quiescent culture has a large amount of sea-tangle juvenile sporophyte to occur for 7 ~ 10 days afterwards as seen, now can start to change water or flowing water, later stage condition of culture is produced with conventional seedbed system, laminaria production seedling can be obtained through the cultivation of 1 ~ 2 month.
8. a kind of kelp seedling breeding method according to claim 7, is characterized in that: the method preparing described N/P nutrient solution: take 121 grams of sodium nitrate, and after dissolving with sterilizing seawater, then continuation sterilizing seawater is settled to 500 ml and obtains sodium nitrate solution; Take 17.5 grams of potassium dihydrogen phosphates, after dissolving with sterilizing seawater, then be settled to 1000 ml with sterilizing seawater and obtain potassium dihydrogen phosphate; Measure potassium dihydrogen phosphate described in sodium nitrate solution described in 1 ml and 1 ml respectively, be added in the sterilizing seawater of 4000 ml, shake up and can obtain described N/P nutrient solution.
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| CN111448987A (en) * | 2020-04-28 | 2020-07-28 | 山东东方海洋科技股份有限公司 | Tissue culture method for obtaining kelp filament and application thereof |
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| CN110249995A (en) * | 2019-07-30 | 2019-09-20 | 集美大学 | A method of long-term preservation pure laver line germ plasm resource at normal temperature |
| CN111448987A (en) * | 2020-04-28 | 2020-07-28 | 山东东方海洋科技股份有限公司 | Tissue culture method for obtaining kelp filament and application thereof |
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