CN104920201B - A kind of kelp seedling breeding method - Google Patents
A kind of kelp seedling breeding method Download PDFInfo
- Publication number
- CN104920201B CN104920201B CN201510354023.7A CN201510354023A CN104920201B CN 104920201 B CN104920201 B CN 104920201B CN 201510354023 A CN201510354023 A CN 201510354023A CN 104920201 B CN104920201 B CN 104920201B
- Authority
- CN
- China
- Prior art keywords
- sea
- tangle
- callus
- cultivation
- children
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
Landscapes
- Breeding Of Plants And Reproduction By Means Of Culturing (AREA)
- Cultivation Of Seaweed (AREA)
Abstract
The present invention discloses a kind of kelp germplasm and preserves and its offspring seed cultivation method, and kelp germplasm store method includes：Sea-tangle children's spore explant or the pretreatment of sea-tangle megaspore explant, the induction of callus, the preservation of callus；Kelp seedling breeding method includes：The spreading cultivation of callus, the seedling differentiation of callus.Sea-tangle explant callus preserving seed of the present invention and its larval rearing are not affected by season and weather, plant fast breeding purpose can just be reached in short time, and it is pure to produce kind of property, quality better, the high productivity sea-tangle seedling of yield, the present invention is not limited by ripe sporinite, the sporinite growing part of each growth period can induce callus as explant, the callus of acquisition can be used as germplasm materials and be preserved for a long time, and can further spread cultivation and break up, produce for breeding and nursery, the present invention has strong applicability, it is easy to manually control, draw materials few, efficiency high, it is simple to operate, it is easy to the characteristics of promoting.
Description
Technical field
The present invention relates to the kelp germplasm of preserving seed and its offspring seed cultivation method, more particularly, to breeding production is preserved
And its offspring seed cultivation method, belong to marine alga tissue cultures and applied technical field.
Background technology
Sea-tangle is large-scale sea-plant, has larger economic worth.Belong to Phaeophyta, brown son in natural classification system
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 in seabed more
It is on cay, individual larger, it is that artificial cultivation produces most marine algas.Sea-tangle is by large-scale sporinite (i.e. commodity life throughout one's life
Produce object " sea-tangle ") it is alternate with each other with small gametophytic generation from generation to generation and constitute.The big individuality of sporinite is tame
Object, accounts for the time in the history of life long.Gametophytic generation is the object of artificial culture seed, and now production is all constant temperature stream indoors
Carry out under water condition.Sea tangle sporophyte is divided into blade, three parts of handle and holdfast.The frond blade of growth be in banding, nothing
Branch, brown and be rich in gloss.There are two shallow ridges to pass through " middle band portion " is formed in middle part of blade.Blade base and the ground of handle connection
Side is the growing part of frond, sea-tangle of the length more than one meter, and the position of growing part is from blade base to away from 10 centimetres of base portion
The position of left and right, the cell division function at this position are very strong.In sea-tangle growth course, growing part cell constantly divides, and makes new group
Knit and constantly increase from blade base, and old tissue is then pushed to front, thus from blade base more away from tissue its formed when
Between it is also more early, thus aging is also early, starts at first to degrade, and this growth pattern of sea-tangle is grown between being just called.This inter-species grows
Algae, its separate living tissue be located at blade base portion, in growth course, new organization constantly from leaf base increase, to ending
Passage, show the growth of algae, while taper gradually aging comes off.The purpose of higher plant tissue cultures is to obtain complete
Plant, because being soil-less culture, with solid medium to play a part of fixed plant, and sea-tangle belongs to aquatic ocean
Plant, cultivates in being totally submerged liquid medium within, and fluid nutrient medium nutrient content is uniform, so inoculum density is than solid
Body culture medium can be with much larger, and such yield is also big.
The callus concept of brown alga meets higher plant, and this callus gives appropraite condition, just can break up again,
Develop into normal sporinite；Can also preserve for a long time；Can also be cultivated by suspending and be bred rapidly, be carried out for use as clone
Nursery is produced.The research of brown alga callus starts from saga (Saga, N., T.Uchida＆Y.Sakai, 1978.Clone in 1978
Laminaria from single isolated cell.Bull.jap.Soc.sci.Fish.44:87.) sea-tangle is studied, he
After callus is obtained, individual cells are separated to from callus, and induce into sporinite, demonstrate brown alga callus first
Histiocytic totipotency.Yan Zuomei (marine products institute of Chinese Marine University retirement professor, Fang Zongxi, Yan Zuomei, Wang Zongcheng；Sea
The preliminary observation [J] of band and undaria pinnitafida tissue cultures；Science Bulletin；11 phase of nineteen eighty-two) in 1984 once to sea-tangle and undaria pinnitafida
Different tissues make comparisons, find blade base (growing point) and middle with portion's formation callus and the maximum probability that breaks up again, shank
Take second place, rhizoid portion is worst, and rear two parts are difficult to be divided into sporinite again.(1967.3-, the Chinese Academy of Sciences ocean such as Wang Xihua
Research institute assistant researcher, Wang Xihua, Qin Song, Zeng Chengkui；High efficiency induction [J] of sea-tangle callus；Oceanologia et Limnologia Sinica；
06 phase in 1999) callus was obtained using solid medium induction sea tangle sporophyte in 1999, but have no callus
Spread cultivation, break up and plant regeneration relevant report.
Marine alga Study on tissue culture is in progress from the sixties in last century, after the eighties relatively rapidly, Zhao Huandeng and Zhang Xue
Polishing, Lu Chengqing (nineteen eighty-three) methods of rotting, Saga (1984) is used to be separated to Porphyra yezoensis list with enzyme process into (1981)
Cell simultaneously all regenerates strain.The country undertaken by the Institute of Oceanology of the Chinese Academy of Sciences, Shanghai Aquatic Products Univ. 9CN) and Qingdao Marine University
" the Seventh Five-Year Plan " brainstorm subject " laver cell nursery " is in nineteen ninety by checking and accepting.Expert thinks that the problem utilizes cell culture skill
Art carries out nursery, greatly shortens seedling raise period than conventional method, and seedling rearing room floor space is little, low cost, with certain society
And economic benefit, reach the international leading level.Marine alga tissue cultures develop in seaweed it is more ripe, and in sea-tangle and undaria pinnitafida etc.
Research in China Main Economic marine alga is deep not enough.
Current kelp germplasm is preserved and breeding, raising seedling mainly has two kinds of approach：One kind is by Dai Xuan using ripe sporinite
Plant, the mode of nursery carries out conservation, fine-variety breeding and nursery are produced, this conservation and Seedling production mode are in the mistake selected by generation
The loss and degeneration of breeding merit are inevitably resulted in journey, and breeding of new variety are carried out with the method and then exist educating
Kind of cycle length, Breeding Efficiency are low, the drawbacks of high labor intensive.Another kind of approach is based on gametophytic conservation and breeding, raising seedling side
Formula.This mode diffuses trip spore by ripe sporinite, when the male and female gametophytes that can be offered an explanation under the microscope are developed into,
By its single long-term preservation, breeding and nursery production for separating and preserving, cultivate, can be used for germplasm.This mode has compared with the former
Season and sea area condition are not limited and the advantages of breeding cycle is shorter for variety height, long service life, nursery.Both the above
Premised on sea-tangle conservation, breeding and nursery approach have sporangial ripe sporinite by length, and for by unstable mode
Some phenotypic characters that (such as radiation, mutagenesis or natural mutation etc.) or alternate manner are obtained are projected but do not grow sporangial
Body, there is no effective preserving seed and offspring seed cultivation method at present.
The content of the invention
The technical problem to be solved in the present invention is：There is provided it is a kind of using sea-tangle explant callus carry out preserving seed and
The method of its larval rearing.
The present invention technical solution be：The method that the first kelp germplasm is preserved, its step is：
(1) pretreatment of sea-tangle children spore explant：
After a, the sterilized seawater flushing of sea-tangle juvenile sporophyte, with holdfast and shank of the blade sea-tangle juvenile sporophyte of sterilizing
Cut off and obtain sea-tangle children's spore stripping and slicing, then remove sea-tangle children's spore diced facets debris, and it is young with sterilizing seawater flushing sea-tangle
Spore stripping and slicing,
B, sea-tangle children's spore stripping and slicing is moved in superclean bench, with the liquor kalii iodide that concentration is 1.5% at twice first
Soak, every time 7～10 minutes sterilizings, then move in dual anti-solution, also soak at twice, sterilization in 7～10 minutes every time disappears
Poison, then use sterilizing seawater flushing 2～3 times,
C, with sterilizing blade by sea-tangle children's spore stripping and slicing by base portion, the 1/4～1/5 of the children's spore stripping and slicing of whole sea-tangle cuts
Under, sea-tangle children's spore growth portion's stripping and slicing is obtained, then sea-tangle children's spore growth portion's stripping and slicing edge trimming is made into sea-tangle children with sterilizing knife
New section exposes in stripping and slicing outward flange in spore growth portion, and sea-tangle children's spore growth portion's stripping and slicing that new section is exposed in outward flange is cut into again
Area is 2～3mm2Bulk obtain sea-tangle children's spore explant；
(2) induction of callus：Sea-tangle children's spore explant is placed in culture dish, adds concentration to be 2%
PESI nutrient solutions, are then cultivated in illumination box, and cultivation temperature is 15 DEG C, and intensity of illumination is 1500～2000lux,
Photoperiod is 10L:14D, the PESI nutrient solutions were changed once per 7 days, and sea-tangle children's spore explant Jing is cultivated for 1～2 month, is obtained
To sea-tangle callus；
(3) preservation of callus：By the sea-tangle callus turned out sterilizing blade by sea-tangle children's spore explant
Cut and collect, proceed to 100ml conical flasks and cultivate in illumination box, culture medium is cultivated for the PESI that concentration is 2%
Liquid, cultivation temperature are 5～10 DEG C, and intensity of illumination is 500～1000lux, and the photoperiod is 10L:14D, the PESI nutrient solutions are every
Change once within 30 days, the sea-tangle callus for obtaining with this understanding can be preserved for a long time as kelp germplasm material.
The method that second kelp germplasm of the present invention is preserved, its step is：
(1) pretreatment of sea-tangle megaspore explant：
A, sea-tangle macrosporinite are once purged, sea-tangle macrosporinite growing part is cut and obtains sea-tangle macrosporinite growing part
Stripping and slicing, remove sea-tangle macrosporinite growing part diced facets debris, and with sterilize seawater flushing,
B, by the stripping and slicing of sea-tangle macrosporinite growing part move into superclean bench in, it is first molten with the KI that concentration is 1.5%
Liquid soaks at twice, every time 7～10 minutes sterilizings, then moves in dual anti-solution, also soaks at twice, 7～10 points every time
Clock sterilizing, then use sterilizing seawater flushing 2～3 times,
C, by sea-tangle macrosporinite growing part stripping and slicing edge trimming, expose sea-tangle macrosporinite growing part stripping and slicing outward flange
The sea-tangle macrosporinite growing part stripping and slicing that new section is exposed in outward flange is cut into area for 2～3mm by new section again2It is block
To sea-tangle megaspore explant；
(2) induction of callus：Sea-tangle megaspore explant is placed in culture dish, macrosporinite culture is added
Liquid, macrosporinite nutrient solution are the mixed liquor containing PESI and NAA, and the concentration of the PESI in mixed liquor is 2%, NAA final concentrations
For 1～1.86mg/L, then cultivated in illumination box, cultivation temperature be 15 DEG C, intensity of illumination be 1500～
2000lux, photoperiod are 10L:14D, mixed liquor were changed once per 7 days, and sea-tangle megaspore explant Jing is cultivated for 1～2 month, is obtained
To sea-tangle callus；
(3) preservation of callus：The sea-tangle callus turned out is sterilized into blade by sea-tangle megaspore explant
On cut and collect, proceed to 100ml conical flasks and cultivate in illumination box, culture medium is PESI trainings that concentration is 2%
Nutrient solution, cultivation temperature are 5～10 DEG C, and intensity of illumination is 500～1000lux, and the photoperiod is 10L:14D, PESI nutrient solution is per 30
Once, the sea-tangle callus for obtaining with this understanding can be preserved for a long time as kelp germplasm material for its replacing.
The Compound mixed solution method of the PESI and NAA of above-mentioned steps (2)：10mg methyl α-naphthyl acetates are weighed, with 500 μ l concentration is first
The sodium hydroxide solution dissolving of 1mol/l, adds distilled water and is settled to 10ml, then sterilized with 0.22 μm of filter membrane suction filtration
To the naphthalene acid solution that concentration is 1mg/ml；Again the naphthalene acid solution is added in the PESI nutrient solutions that concentration is 2%, often
100ml concentration be 2% PESI nutrient solutions in plus concentration 1mg/ml 100～186 μ l of methyl α-naphthyl acetate.
Above-mentioned concentration is that the collocation method of 2% PESI nutrient solutions is：Weigh the phosphoglycerol of 350mg sodium nitrate, 50mg
Sodium, the iron edta sodium salt of 2.5mg, the trishydroxymethylaminomethane of 500mg, the KI of 100 μ g, measure 25ml's
II molten metals of P- add the sodium nitrate, sodium glycero-phosphate, iron edta sodium salt, trishydroxymethylaminomethane, iodate
After potassium dissolving, 100ml is settled to distilled water, adjustment pH value is the 7.8 PESI nutrient solutions for obtaining that concentration is 2%, the P-
The collocation method of II molten metal is：Weigh the disodium ethylene diamine tetraacetate of 100mg, the iron chloride of 1mg, the boric acid of 20mg, 4mg
Manganese chloride, the zinc chloride of 0.5mg, the cobalt chloride of 0.1mg, with distillation water dissolves, and are settled to 100ml with distilled water.
Above-mentioned concentration is the collocation method of 1.5% liquor kalii iodide：1.5g KIs are weighed, with sterilizing seawater dissolving
Afterwards, and 100ml is settled to sterilizing seawater obtain the liquor kalii iodide that concentration is 1.5%；The configuration side of the dual anti-solution
Method：0.08g Ciprofloxacin Lactates, 0.1g penicillin are weighed, and with distillation water dissolves, and 1000ml are settled to distilled water and are obtained
Dual anti-solution.
Above-mentioned sterilizing blade is adopted in pressure as 103.4kPa, and temperature is sterilizing 30 minutes under conditions of 121 DEG C.
Kelp seedling breeding method of the present invention, its step is：
(1) callus spreads cultivation：The sea-tangle callus of above-mentioned acquisition is shredded, the PESI trainings that concentration is 2% are proceeded to
In nutrient solution, cultivation temperature is 15 DEG C, and intensity of illumination is 2000～3000lux, and the photoperiod is 10L:14D, the PESI nutrient solutions
Changed once per 7 days, through the culture of 20～30 days, sea-tangle callus was grown up；Again collect the sea-tangle callus group after growing up
Knit and shred, then cultivated in proceeding to the PESI nutrient solutions that concentration is 2% again, through the culture of 20～30 days, sea-tangle callus group
Knit and grow up；Above operation is continuously repeated, sea-tangle callus can keep vigorous growth, the callus after being spread cultivation；
(2) seedling differentiation of callus：Collect the callus after spreading cultivation and smash, change the PESI that concentration is 2%
Nutrient solution carries out renewal cultivation, and the temperature of renewal cultivation is 15 DEG C, and intensity of illumination is 2000～3000lux, and the photoperiod is 10L:
14D, callus renewal cultivation were proceeded in low temperature nursery storehouse, are inoculated on breeding screen, with fluorescent lamp or nature after one week
Light is light source, and initial intensity of illumination is 2000～2500lux, and water temperature is 8～10 DEG C, and nutrient solution is replaced by N/P nutrient solutions, is stood
It is visible after cultivating 7～10 days to have a large amount of juvenile sporophytes to occur, can now start to change water or flowing water, later stage condition of culture is educated with conventional
Seedling is produced, and laminaria production seedling is obtained through the culture of 1～2 month.
The method for preparing above-mentioned N/P nutrient solutions：121 grams of sodium nitrate are weighed, after being dissolved with sterilizing seawater, is further continued for sterilizing
Seawater is settled to 500ml and obtains sodium nitrate solution；Weigh 17.5 grams of potassium dihydrogen phosphates, with sterilizing seawater dissolving after, then with sterilize
Seawater is settled to 1000ml and obtains potassium dihydrogen phosphate；Biphosphate described in sodium nitrate solution described in 1ml and 1ml is measured respectively
Potassium solution, is added in the sterilizing seawater of 4000ml, shakes up.
The solution have the advantages that：Present invention fluid nutrient medium induction kelp spore explant is (outside sea-tangle children's spore
Implant and sea-tangle megaspore explant) can obtain sea-tangle callus high efficiency induction；Certain condition of culture is given, more
Injured tissue can be used as germplasm materials and be preserved for a long time, and callus is spread cultivation and broken up, and can cultivate substantial amounts of sea
Band seedling, these kelp seedlings can be used for breeding production.
Sea-tangle explant callus preserving seed of the present invention and its larval rearing are not affected by season and weather, in short-term
It is interior just to reach plant fast breeding purpose, and the high productivity sea-tangle use of pure kind of property, quality better, yield can be produced
Seedling.Research shows, in the plant living cells of ex vivo situation, in the work of suitable nutriment, hormone and other external conditions
With under, it is possible to show totipotency, complete plant is developed into.The present invention is not limited by ripe sporinite, each growth period
Sporinite growing part can induce callus as explant, the callus of acquisition can be used as germplasm materials and enter
Row is long-term to be preserved, and can further spread cultivation and break up, and produces for breeding and nursery.The present invention has strong applicability, is easy to people
Industry control system, draw materials less, efficiency high, it is simple to operate, be easy to promote the characteristics of, be kelp germplasm preserve and breeding, raising seedling open one
The new approach of bar, while alternatively cytology and genetics research provide material, is being carried out using sea-tangle tissue and cell cultivation
Also there is in terms of the research that secondary substance synthesizes and specific drugs are converted potential using value.
Specific embodiment
It is described in detail with reference to specific embodiment：
Embodiment 1
Embodiment 1 be carry out pre-processing using sea-tangle juvenile sporophyte (kelp seedling), the induction of callus and preservation, then
Spread cultivation by callus and seedling differentiation.
(1) pretreatment of sea-tangle children spore explant：Select the complete hurtless measure of blade, healthy and bright in colour, miscellaneous algae dirty
The few sea-tangle juvenile sporophyte of dye, length is between 1.0～2.5cm.After the sterilized seawater of sea-tangle juvenile sporophyte is repeatedly rinsed, with sterilizing
Blade cuts off sea-tangle juvenile sporophyte holdfast and shank；Gently wipe sea-tangle juvenile sporophyte blade surface to go removal of impurities with cotton swab
Thing, and with sterilizing seawater flushing, repeatable to wipe several standby after rinsing again, when noting wiping, dynamics gently will should not damage sea
Band juvenile sporophyte blade.
The sea-tangle juvenile sporophyte blade for having wiped with the alcohol disinfecting that concentration is 75%, is then moved by operator's both hands in advance
Enter in the superclean bench of prior ultraviolet disinfection, first with the liquor kalii iodide (KI) that concentration is 1.5%, total immersion steeps 15min at twice
Sterilizing, then move in dual anti-solution, also soak at twice, coprocessing 15min, then use sterilizing seawater flushing 2～3 times,
The dual anti-solution of residual is rinsed out.With sterilizing blade by sea-tangle juvenile sporophyte blade by base portion, whole blade 1/4～
1/5 cuts and obtains sea-tangle juvenile sporophyte growing part stripping and slicing, that is, cut the growing part that material is sea-tangle juvenile sporophyte.The present embodiment sea
Band juvenile sporophyte liquor kalii iodide soaks at twice, every time 7～10 minutes sterilizings, then sea-tangle juvenile sporophyte is moved into double
In anti-solution, also soak at twice, 7～10 minutes every time.
Take sea-tangle juvenile sporophyte growing part stripping and slicing, with sterilizing knife by four edges of sea-tangle juvenile sporophyte growing part stripping and slicing again
Cut away a bit, expose new section, after 2～3mm is cut into in the sea-tangle juvenile sporophyte growing part stripping and slicing of reservation again2Bulk as sea
The material of tissue cultures is done with young spore explant.
(2) induction of callus：By sea-tangle children's spore explant culture in the glass culture dish of a diameter of 9cm, training
Add the PESI nutrient solution 30ml that concentration is 2% in foster ware in advance, then 7～8 pieces of sea-tangle children's spore explants are put into tweezers
In nutrient solution；The glass culture dish for being placed with PESI nutrient solutions and sea-tangle children's spore explant is trained in illumination box
Support, cultivation temperature is 15 DEG C, and intensity of illumination is 1500～2000lux, and the photoperiod is 10L:14D (10 hours of illumination, dark 14
Individual hour, circulation conversion), PESI nutrient solutions were changed once per 7 days, and each full dose is changed, and carries out structure observation.Sea-tangle children's spore
Sub- explant Jing is cultivated for 1～2 month, can obtain sea-tangle callus.
(3) preservation of callus：By the sea-tangle callus turned out sterilizing blade by sea-tangle children's spore explant
Cut and collect, proceed to 100ml conical flasks and cultivate in illumination box, culture medium is cultivated for the PESI that concentration is 2%
Liquid, cultivation temperature are 5～10 DEG C, and intensity of illumination is 500～1000lux, and the photoperiod is 10L:14D, PESI nutrient solution was per 30 days
Change once, each full dose is changed.With this understanding, sea-tangle callus growth is slower, can be long as germplasm materials
Phase preserves.
(4) callus spreads cultivation：Sea-tangle callus scalpel is shredded or high-speed tissue mashing machine smashes, proceeded to
During fresh concentration is 2% PESI nutrient solutions, cultivate in illumination box, cultivation temperature is 15 DEG C, intensity of illumination is
2000～3000lux, photoperiod are 10L:14D, nutrient solution are changed once for 7 days, and each full dose is changed.Through about 15～20 days
Culture, sea-tangle callus are grown up, and can collect again sea-tangle callus and be shredded or high-speed tissue mashing machine with scalpel again
Smash, then proceed in the PESI nutrient solutions that fresh concentration is 2%, cultivate in illumination box, cultivation temperature is 15 DEG C, light
It is 2000～3000lux according to intensity, the photoperiod is 10L:14D, nutrient solution 7 days are changed once, then through the culture of 15～20 days,
Above operation is repeated, such sea-tangle callus can keep vigorous growth, you can reach the purpose of Amplification Culture, in case subsequently
Nursery is used.
(5) seedling differentiation of callus
After sea-tangle callus spreads cultivation to requirement, collected and fully smashed with high-speed tissue mashing machine,
And the concentration for more renewing is 2% PESI nutrient solutions, renewal cultivation was proceeded in low temperature nursery storehouse, is inoculated in and is educated after one week
On seedling curtain, with fluorescent lamp or natural light as light source, initial intensity of illumination is 2000～2500lux, and water temperature is 8～10 DEG C, culture
Fluid exchange is N/P nutrient solutions, and quiescent culture is visible after 7～10 days to have a large amount of juvenile sporophytes to occur, and at this moment can start periodic replacement
New nutrient solution carries out flowing water culture, and later stage condition of culture is with routine kelp seedling cultivation production, the training of conventional kelp seedling cultivation production
Educate water temperature：7～8 DEG C.Intensity of illumination：With natural light as light source, manual adjustment intensity of illumination, sea-tangle juvenile sporophyte length are less than
During 0.2mm, highest light intensity 3500lux, 1700～1800lux of average intensity；Sea-tangle juvenile sporophyte length in 0.2～0.3mm,
Highest light intensity 4000lux, 1800～2000lux of average intensity；Sea-tangle juvenile sporophyte length in 0.3～3mm, highest light intensity
4000lux, 2000～2200lux of average intensity；Sea-tangle juvenile sporophyte length in 3～5mm, highest light intensity 5000lux, averagely
2200～2500lux of light intensity；Sea-tangle juvenile sporophyte length in 5～10mm, highest light intensity 5500lux, average intensity 2500～
2800lux；Sea-tangle juvenile sporophyte length in more than 1cm more than highest light intensity 6000lux, more than average intensity 3000lux.Day
Quantity of exchanged water：Before sea-tangle juvenile sporophyte length reaches 5mm, daily quantity of exchanged water is the 50% of total water body；Sea-tangle juvenile sporophyte length reaches
To after 5mm, daily quantity of exchanged water is the 75% of total water body；After sea-tangle juvenile sporophyte length reaches 10mm, daily quantity of exchanged water is total water
The 100% of body.Clear pond：To avoid miscellaneous algae and bacterium amount reproduction, according to water quality condition and juvenile sporophyte growing state per 10 days
Culture pond is scrubbed thoroughly once in left and right.Flow velocity and flow time：During flowing water culture, front 24～48 hours flow velocitys are less than 0.05m/s,
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, sea-tangle children before 0.2mm
Spore body length scrubs initial pressure 0.5KG in 0.2～0.3mm, scrubs pressure and is gradually increased 1.0KG；Sea-tangle children's spore
Body length scrubs pressure for 1.5KG in 0.3～3mm；In 3～5mm, scrub pressure is sea-tangle juvenile sporophyte length
2.0KG；Sea-tangle juvenile sporophyte length scrubs pressure maximum less than 2.5KG in more than 5mm.Culture through 1～2 month is
The production kelp seedling of 1～2cm of length is obtained.
Embodiment 2
Embodiment 2 be carry out pre-processing using sea-tangle macrosporinite (adult sea-tangle), the induction of callus and preservation, then
Spread cultivation by callus and seedling differentiation.
(1) pretreatment of explant：Select the complete hurtless measure of blade, the healthy and bright in colour, sea-tangle that miscellaneous algae pollution is few
Macrosporinite (adult sea-tangle) is material.After sea-tangle is at sea tentatively cleaned by sea-tangle macrosporinite with seawater, laboratory is taken
Sea-tangle macrosporinite growing part is cut with scissors, sea-tangle macrosporinite growing part is wiped repeatedly with absorbent cotton, to remove sea-tangle
The debris on macrosporinite growing part surface, and it is standby after being rinsed with sterilizing seawater repeatedly.
Operator's both hands concentration is 75% alcohol disinfecting, the sea-tangle macrosporinite growing part for having wiped is moved into purple in advance
In the superclean bench of outer sterilization, first with KI (KI) solution that concentration is 1.5%, the sterilization in 15 minutes of total immersion bubble disappears at twice
Poison, then move in dual anti-solution, also soaks, coprocessing 15 minutes at twice, with sterilizing seawater flushing 2～3 times, by the double of residual
Anti- solution is rinsed out.
Four edges of sea-tangle macrosporinite growing part are cut away one with scalpel by sea-tangle macrosporinite growing part stripping and slicing again
Point, exposes new section, after the stripping and slicing for exposing new section is cut into into area again for 2～3mm2Block sea-tangle megaspore explant do
The material of tissue cultures.
(2) induction of callus：By sea-tangle megaspore explant culture in the glass culture dish of a diameter of 9cm, sea
It is the mixed liquor containing PESI and NAA (methyl α-naphthyl acetate) with megaspore explant nutrient solution, the concentration of the PESI in mixed liquor is
Final concentration of 1～the 1.86mg/L of 2%, NAA,；Each glass culture dish adds PESI and NAA mixed liquors about 30ml, then places into 7
～8 pieces of sea-tangle megaspore explants；Cultivated in illumination box, cultivation temperature be 15 DEG C, intensity of illumination be 1500～
2000lux, photoperiod are 10L:14D, nutrient solution were changed once per 7 days, and each full dose is changed, and carries out structure observation.Sea-tangle
Megaspore explant Jing is cultivated for 1～2 month, you can obtain sea-tangle callus.
(3) preservation of callus：The sea-tangle callus turned out is sterilized into blade by sea-tangle megaspore explant
On cut and collect, proceed to 100ml conical flasks and cultivate in illumination box, culture medium is PESI trainings that concentration is 2%
Nutrient solution, cultivation temperature are 5～10 DEG C, and intensity of illumination is 500～1000lux, and the photoperiod is 10L:14D, nutrient solution per 30 days more
Change once, each full dose is changed.With this understanding, sea-tangle callus growth is slower, can carry out for a long time as germplasm materials
Preserve.
(4) callus spreads cultivation：Sea-tangle callus scalpel is shredded or smashed with high-speed tissue mashing machine, is turned
Enter in the PESI nutrient solutions that fresh concentration is 2%, cultivation temperature be 15 DEG C, intensity of illumination be 2000～3000lux, the photoperiod
For 10L:14D, nutrient solution were changed once per 7 days, and each full dose is changed.Through the culture of about 20～30 days, callus was grown up,
Can collect again and be shredded with scalpel or high-speed tissue mashing machine smashes, proceed to the PESI nutrient solutions that fresh concentration is 2%
Middle culture, then through the culture of 20～30 days, above operation is repeated, such sea-tangle callus can keep vigorous growth, i.e.,
The purpose of Amplification Culture is can reach, in case follow-up nursery is used.
(5) seedling differentiation of callus
(spread cultivation to can be general The more the better with the amount of nursery production, with not when sea-tangle callus spreads cultivation to requirement
It is complete to continue long-term preservation) after, the sea-tangle callus after spreading cultivation is collected fully to be smashed with high-speed tissue mashing machine,
And the concentration for more renewing is 2% PESI nutrient solutions, renewal cultivation was proceeded in low temperature nursery storehouse, is inoculated in and is educated after one week
On seedling curtain, with fluorescent lamp or natural light as light source, initial intensity of illumination is 2000～2500lux, and water temperature is 8～10 DEG C, culture
Fluid exchange is N/P nutrient solutions, and quiescent culture is visible after 7～10 days to have a large amount of sea-tangle juvenile sporophytes to occur, and can now start to change water
Or flowing water, later stage condition of culture is with conventional seedbed system production.Culture through 1～2 month is obtained the production of 1～2 centimetre of length
Use kelp seedling.
Cotton swab, absorbent cotton, glass culture dish, sterilizing blade, tweezers, scalpel and high-speed tissue mashing machine used by embodiment
Autoclaving will be shifted to an earlier date with vertical pressure steam sterilization pan Deng apparatus, auxiliary material, sterilization pressure is 103.4kPa, sterilising temp is
121 DEG C, sterilization time 30 minutes.
The embodiment of the present invention adopts following laboratory apparatus：
A, illumination box：MGC-300A types, one permanent Science and Technology Ltd. of Shanghai
B, microscope：CX22 types, OLYMPUS
C, refrigerator：BCD-216TX types, Qingdao HaiEr Co., Ltd
D, high-speed tissue mashing machine：DS-1 types, Shanghai Sample Model Factory
E, superclean bench：SW-CJ-ID types, 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) the seawater seawater of making a living that sterilizes boils 2 minutes, and naturally cools to room temperature.
2) concentration is 1.5% liquor kalii iodide：Weigh 1.5g KIs (molecular formula KI, the chemistry examination of Shanghai Chinese medicines group
Agent Co., Ltd produces, and analyzes pure), dissolved with sterilizing seawater and be settled to 100ml.
3) dual anti-solution：(molecular formula is C to weigh 0.08g Ciprofloxacin Lactate soluble powders17H18FN3O3, Guangdong Ke Run
Biology Pharmacy Co., Ltd produces), (molecular formula is C to 0.1g penicillin16H17N2NaO4S, Lukang Medical Co., Ltd., Shandong
Production, 800,000 units), water dissolves 1000ml is settled to distillation.
4) concentration is 2% PESI nutrient solutions：(molecular formula is NaNO to weigh 350mg sodium nitrate3, Shanghai Chinese medicines group
The production of reagent Co., Ltd is learned, is analyzed pure), (molecular formula is Na to 50mg sodium glycero-phosphates2C3H5(OH)2PO4·5H2O, Chengdu section
Imperial chemical reagent factory production, analyzes pure), (molecular formula is C to 2.5mg iron edta sodium salts10H12FeN2Na0, MDBio,
Inc. produce, analyze pure), (molecular formula is C to 500mg trishydroxymethylaminomethanes4H11NO3, abbreviation is Tris, MDBio, Inc.
Production, analyzes pure), 100 μ g KIs (molecular formula is KI, and the production of Shanghai Chemical Reagent Co., Ltd., Sinopharm Group, analysis are pure),
Measure 25ml II molten metals of P- add dissolving after, be settled to 100ml with distilled water, and with the NaOH that concentration is 1mol/l
Solution adjustment pH value is 7.8.When adjusting pH value with the sodium hydroxide solution that concentration is 1mol/l, when drop sodium hydroxide solution is added
PH is surveyed with pH meter, until solution adjustment pH value is 7.8.
5) II molten metals of P-：(molecular formula is C to weigh 100mg disodium ethylene diamine tetraacetates10H14N2O8Na2·2H2O, Shanghai
Chemical Reagent Co., Ltd., Sinopharm Group produces, and analyzes pure), (molecular formula is FeCl to 1mg iron chloride3, Shanghai Chinese medicines group chemistry
Reagent Co., Ltd produces, and analyzes pure), (molecular formula is H to 20mg boric acid3BO3, the life of Shanghai Chemical Reagent Co., Ltd., Sinopharm Group
Produce, analyze pure), (molecular formula is MnCl to 4mg manganese chlorides2, the production of Shanghai Chemical Reagent Co., Ltd., Sinopharm Group, analysis are pure),
(molecular formula is ZnCl to 0.5mg zinc chloride2, the production of Shanghai Chemical Reagent Co., Ltd., Sinopharm Group, analysis are pure), 0.1mg chlorinations
(molecular formula is CoCl to cobalt2, the production of Shanghai Chemical Reagent Co., Ltd., Sinopharm Group analyzes pure), with distillation water dissolves constant volume
To 100ml.
6) methyl α-naphthyl acetate (NAA) of the compound concentration for 1mg/ml：Weigh 10mg methyl α-naphthyl acetate powder (molecular formula abbreviation NAA, Beijing
Suo Laibao Science and Technology Ltd.s produce), first with the sodium hydroxide solution dissolving that 500 μ l concentration are 1mol/l, add distilled water
10ml is settled to, is sterilized with 0.22 μm of filter membrane suction filtration.This solution is used to be added in 2%PESI nutrient solutions, per 100ml concentration
For 100～186 μ l (microlitre) of methyl α-naphthyl acetate for adding concentration 1mg/ml in 2% PESI nutrient solutions.
7) sodium hydroxide solution of the compound concentration for 1mol/l：Weigh 4g sodium hydroxide powders (molecular formula NaOH, Shanghai state
Chemical reagent Co., Ltd of medicine group produces, and analyzes pure), with distilling water dissolves and being settled to 100ml, for preparing 1mg/ml's
Methyl α-naphthyl acetate.
8) prepare sodium nitrate solution：Weigh 121 grams of sodium nitrate (molecular formula NaNO3, Shanghai Chinese medicines group chemical reagent is limited
Company produces, and analyzes pure), 500ml is dissolved and is settled to sterilizing seawater, for preparing nutrient solution containing N/P.
9) prepare potassium dihydrogen phosphate：Weigh 17.5 grams of potassium dihydrogen phosphate (molecular formula KH2PO4, Shanghai Chinese medicines group
The production of reagent Co., Ltd is learned, is analyzed pure), 1000ml is dissolved and is settled to sterilizing seawater, for preparing N/P nutrient solutions.
10) prepare nutrient solution containing N/P (nitrogen phosphorus nutrient solution)：Above-mentioned 1ml sodium nitrate solutions and 1ml biphosphates are measured respectively
Potassium solution, is added to 4000ml Jing and boils 2 minutes and naturally cool in the sterilizing seawater of room temperature, shake up standby.Plant group
Knit in culture, various organs, tissue and the cell for using is referred to as explant.Callus refer to explant because it is injured or from
During body culture, one kind that its undifferentiated cell and differentiated cell carry out active division growth and formed is without ad hoc structure
With the tissue of function.Callus cell can be preserved, cultivated and be expanded, at the same cell have through dedifferentiation it is all-round
Property, can under proper culture conditions through being differentiated to form new plant again.So through the induction of callus, cell
Amplification, then break up, it is possible to reach the purpose of plant fast asexual propagation.
Claims (3)
1. a kind of kelp seedling breeding method, its step is：
(1) callus spreads cultivation：Sea-tangle callus is shredded, is proceeded in the PESI nutrient solutions that concentration is 2%, cultivation temperature
For 15 DEG C, intensity of illumination is 2000～3000lux, and the photoperiod is 10L:14D, the PESI nutrient solutions were changed once per 7 days, Jing
The culture of 20～30 days is crossed, sea-tangle callus is grown up；Again collect the sea-tangle callus after growing up and shred, then proceed to dense
Spend in the PESI nutrient solutions for 2% and cultivate again, through the culture of 20～30 days, sea-tangle callus was grown up again；Continuously repeat
Operate above, sea-tangle callus can keep vigorous growth, the callus after being spread cultivation,
The acquisition methods of the sea-tangle callus：After a, the sterilized seawater flushing of sea-tangle juvenile sporophyte, with sterilizing blade sea
Holdfast with juvenile sporophyte and shank excision obtain sea-tangle children's spore stripping and slicing, then remove sea-tangle children's spore diced facets miscellaneous
Thing, and with sterilizing seawater flushing sea-tangle children's spore stripping and slicing, b, moves into sea-tangle children's spore stripping and slicing in superclean bench, first uses concentration
Liquor kalii iodide for 1.5% soaks at twice, every time 7～10 minutes sterilizings, then moves in dual anti-solution, also at twice
Immersion, 7～10 minutes sterilizings every time, then with sterilizing seawater flushing 2～3 times, c, with sterilizing blade by sea-tangle children's spore
Stripping and slicing is by base portion, the 1/4～1/5 of whole sea-tangle children spore stripping and slicing cuts, and obtains sea-tangle children's spore growth portion's stripping and slicing, then uses
Sea-tangle children's spore growth portion's stripping and slicing edge trimming is made sea-tangle children spore growth portion stripping and slicing outward flange expose new section by sterilizing knife,
Sea-tangle children's spore growth portion's stripping and slicing that new section is exposed in outward flange is cut into into area again for 2～3mm2Bulk obtain sea-tangle children spore
Sub- explant；D, by sea-tangle children spore explant be placed in culture dish, add concentration be 2% PESI nutrient solutions, Ran Hou
Cultivated in illumination box, cultivation temperature is 15 DEG C, and intensity of illumination is 1500～2000lux, and the photoperiod is 10L:14D,
The PESI nutrient solutions were changed once per 7 days, and sea-tangle children's spore explant Jing is cultivated for 1～2 month, obtains sea-tangle callus；
(2) seedling differentiation of callus：Collect the callus after spreading cultivation and smash, change the PESI cultures that concentration is 2%
Liquid carries out renewal cultivation, and the temperature of renewal cultivation is 15 DEG C, and intensity of illumination is 2000～3000lux, and the photoperiod is 10L:14D,
After callus renewal cultivation one week, proceed in low temperature nursery storehouse, be inoculated on breeding screen, with fluorescent lamp or natural light be
Light source, initial intensity of illumination are 2000～2500lux, and water temperature is 8～10 DEG C, and nutrient solution is replaced by N/P nutrient solutions, quiescent culture
It is visible after 7～10 days to have a large amount of sea-tangle juvenile sporophytes to occur, can now start to change water or flowing water, later stage condition of culture is educated with conventional
Seedling is produced, and laminaria production seedling is obtained through the culture of 1～2 month.
2. a kind of kelp seedling breeding method, its step is：
(1) callus spreads cultivation：Sea-tangle callus is shredded, is proceeded in the PESI nutrient solutions that concentration is 2%, cultivation temperature
For 15 DEG C, intensity of illumination is 2000～3000lux, and the photoperiod is 10L:14D, the PESI nutrient solutions were changed once per 7 days, Jing
The culture of 20～30 days is crossed, sea-tangle callus is grown up；Again collect the sea-tangle callus after growing up and shred, then proceed to dense
Spend in the PESI nutrient solutions for 2% and cultivate again, through the culture of 20～30 days, sea-tangle callus was grown up again；Continuously repeat
Operate above, sea-tangle callus can keep vigorous growth, the callus after being spread cultivation,
The acquisition methods of the sea-tangle callus：A, sea-tangle macrosporinite are once purged, and sea-tangle macrosporinite growing part is cut
Under obtain sea-tangle macrosporinite growing part stripping and slicing, remove the debris of sea-tangle macrosporinite growing part diced facets, and extra large with sterilizing
Water rinse, b, by the stripping and slicing of sea-tangle macrosporinite growing part move into superclean bench in, first with the liquor kalii iodide that concentration is 1.5%
Soak at twice, every time 7～10 minutes sterilizings, then move in dual anti-solution, also soak at twice, 7～10 minutes every time
Sterilizing, then with sterilizing seawater flushing 2～3 times, c, by sea-tangle macrosporinite growing part stripping and slicing edge trimming makes sea-tangle big
New section is exposed in sporinite growing part stripping and slicing outward flange, and the sea-tangle macrosporinite growing part stripping and slicing that outward flange is exposed new section is again
Area is cut into for 2～3mm2Bulk obtain sea-tangle megaspore explant；D, sea-tangle megaspore explant is placed on into culture dish
In, macrosporinite nutrient solution is added, macrosporinite nutrient solution is the mixed liquor containing PESI and NAA, the PESI's in mixed liquor
Concentration is the final concentration of 1～1.86mg/L of 2%, NAA, is then cultivated in illumination box, and cultivation temperature is 15 DEG C, light
It is 1500～2000lux according to intensity, the photoperiod is 10L:14D, mixed liquor were changed once per 7 days, sea-tangle megaspore explant Jing 1
Cultivate within～2 months, obtain sea-tangle callus；
(2) seedling differentiation of callus：Collect the callus after spreading cultivation and smash, change the PESI cultures that concentration is 2%
Liquid carries out renewal cultivation, and the temperature of renewal cultivation is 15 DEG C, and intensity of illumination is 2000～3000lux, and the photoperiod is 10L:14D,
After callus renewal cultivation one week, proceed in low temperature nursery storehouse, be inoculated on breeding screen, with fluorescent lamp or natural light be
Light source, initial intensity of illumination are 2000～2500lux, and water temperature is 8～10 DEG C, and nutrient solution is replaced by N/P nutrient solutions, quiescent culture
It is visible after 7～10 days to have a large amount of sea-tangle juvenile sporophytes to occur, can now start to change water or flowing water, later stage condition of culture is educated with conventional
Seedling is produced, and laminaria production seedling is obtained through the culture of 1～2 month.
3. a kind of kelp seedling breeding method according to claim 1 or claim 2, it is characterised in that：Prepare the N/P nutrient solutions
Method：Weigh 121 grams of sodium nitrate, with sterilizing seawater dissolving after, be further continued for being settled to 500ml with sterilizing seawater that to obtain sodium nitrate molten
Liquid；Weigh 17.5 grams of potassium dihydrogen phosphates, with sterilizing seawater dissolving after, then be settled to 1000ml with sterilizing seawater and obtain biphosphate
Potassium solution；Potassium dihydrogen phosphate described in measuring sodium nitrate solution described in 1ml and 1ml respectively, is added to the sterilizing seawater of 4000ml
In, shake up.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201510354023.7A CN104920201B (en) | 2015-06-25 | 2015-06-25 | A kind of kelp seedling breeding method |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201510354023.7A CN104920201B (en) | 2015-06-25 | 2015-06-25 | A kind of kelp seedling breeding method |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN104920201A CN104920201A (en) | 2015-09-23 |
| CN104920201B true CN104920201B (en) | 2017-04-05 |
Family
ID=54107919
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201510354023.7A Active CN104920201B (en) | 2015-06-25 | 2015-06-25 | A kind of kelp seedling breeding method |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN104920201B (en) |
Families Citing this family (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106489708A (en) * | 2016-09-19 | 2017-03-15 | 深圳职业技术学院 | Gracilaria tenuistipitata algae lures method for root |
| CN108849502A (en) * | 2018-06-12 | 2018-11-23 | 金华市胤宏农业科技有限公司 | A kind of cultural method of bladder-wrack |
| CN109694878B (en) * | 2018-12-29 | 2021-05-14 | 中国海洋大学 | Kelp molecule breeding method based on protoplast |
| 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 |
| CN111448987B (en) * | 2020-04-28 | 2022-03-11 | 山东东方海洋科技股份有限公司 | Tissue culture method for obtaining kelp filament and application thereof |
| CN111543305A (en) * | 2020-06-16 | 2020-08-18 | 山东东方海洋科技股份有限公司 | Simple and efficient kelp seedling culture method by improving seedling curtain dark treatment technology |
| CN114503908A (en) * | 2022-03-13 | 2022-05-17 | 威海长青海洋科技股份有限公司 | Kelp seedling culture method for controlling zoospore diffusion through accurate counting |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1373633A (en) * | 2000-08-31 | 2002-10-09 | 科学与工业研究委员会 | Improved process for cultivation of algae |
| CN102771394A (en) * | 2012-07-27 | 2012-11-14 | 山东东方海洋科技股份有限公司 | Method for cloning and culturing seaweed gametophytes |
| CN103858746A (en) * | 2014-03-27 | 2014-06-18 | 青岛崂好人海洋生物技术有限公司 | Seaweed cultivation method |
| CN104542237A (en) * | 2014-12-26 | 2015-04-29 | 中国科学院海洋研究所 | Seedling-raising method for gracilaria algae |
-
2015
- 2015-06-25 CN CN201510354023.7A patent/CN104920201B/en active Active
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1373633A (en) * | 2000-08-31 | 2002-10-09 | 科学与工业研究委员会 | Improved process for cultivation of algae |
| CN102771394A (en) * | 2012-07-27 | 2012-11-14 | 山东东方海洋科技股份有限公司 | Method for cloning and culturing seaweed gametophytes |
| CN103858746A (en) * | 2014-03-27 | 2014-06-18 | 青岛崂好人海洋生物技术有限公司 | Seaweed cultivation method |
| CN104542237A (en) * | 2014-12-26 | 2015-04-29 | 中国科学院海洋研究所 | Seedling-raising method for gracilaria algae |
Non-Patent Citations (2)
| Title |
|---|
| 海带和裙带菜组织培养的初步观察;方宗熙等;《科学通报》;19821130(第11期);第690-691页 * |
| 海带愈伤组织的高效率诱导;王希华等;《海洋与湖沼》;19991130;第30卷(第6期);第652-657页 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN104920201A (en) | 2015-09-23 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN104920201B (en) | A kind of kelp seedling breeding method | |
| CN102907327B (en) | Tissue culture propagation method for anoectochilus roxburghii | |
| CN101999317B (en) | Multiploid bulb induction method with firtillaria cirrhosa scale leaf as explant | |
| CN105706880A (en) | Purple sweet potato virus-free seedling water culture fast breeding method | |
| CN100462001C (en) | Method for fast breeding water caltrop seed and seedling | |
| CN104542237B (en) | Seedling-raising method for gracilaria algae | |
| CN104663439B (en) | Tissue culture and rapid propagation method of waterweed | |
| CN106172008A (en) | A kind of Bowring cattleya fast culture propagation method | |
| CN109042330A (en) | A kind of method for tissue culture of spindle tree | |
| CN102232359B (en) | In-vitro rapid propagation method of double-petal Jasminum sambac | |
| CN105132354A (en) | Extraction method of gracilaria blodgettii protoplast | |
| CN105123512B (en) | A kind of breeding method of roxburgh anoectochilus terminal bud | |
| CN106465680B (en) | Rapid celery tissue culture system | |
| CN104137773B (en) | A kind of method for creating of switchgrass mutant | |
| CN105284622A (en) | Method for rapid acquisition of excellent hybrid clone of iris | |
| CN108633742A (en) | A kind of China fir Stem tip induction culture medium and abductive approach | |
| CN111448987B (en) | Tissue culture method for obtaining kelp filament and application thereof | |
| CN114521489A (en) | Method for inducing willow chromosome doubling at colchicine variable temperature | |
| CN103004592B (en) | Method for breeding, culturing and preserving paratylenchus by using carrot callus | |
| CN106665358A (en) | Rapid shoot induction and tissue culture propagation method for platycodon grandiflorum leaves | |
| CN101422134A (en) | Sargassum thunbergii young seedling large-scale breeding method using leaves tissue culture method | |
| CN111642398A (en) | Control method of endophyte of plant tissue culture seedling | |
| CN105706925B (en) | A kind of method of pale reddish brown cup hat rattan tissue-culturing rapid propagation | |
| CN109699494A (en) | Cultivate the method for adsorbing the sargassum thunbergii of metal ion in waste water | |
| KR100334629B1 (en) | Method for manufacturing high quality young seedling of phalaenopsis in bioreactor by using tissue of flower stalk before blooming |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |