CN1317944C - Wheel leaf black algae engineering seedling fast breeding method - Google Patents
Wheel leaf black algae engineering seedling fast breeding method Download PDFInfo
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- CN1317944C CN1317944C CNB2004100138484A CN200410013848A CN1317944C CN 1317944 C CN1317944 C CN 1317944C CN B2004100138484 A CNB2004100138484 A CN B2004100138484A CN 200410013848 A CN200410013848 A CN 200410013848A CN 1317944 C CN1317944 C CN 1317944C
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- hydrilla verticillata
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A40/00—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production
- Y02A40/80—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production in fisheries management
Abstract
The present invention belongs to the field of a biological technique. High quality engineering seedlings are obtained by a blade hydrilla verticillata tissue culture technique and propagated in a large-scale and expansile mode to overcome problems that seed sources of species are limited in a natural environment, and the species are polluted seriously. The present invention satisfies urgent requirement that every high quality engineering seedling is used as a water purifying tool and a vegetation recovery pioneer species in lake ecological remediation engineering at present, as a place for baits, refuge and spawning in the process of the cultivation of fishes, prawns and crabs in a pond and as a scenery material in the process of the cultivation of indoor ornamental aquatic animals.
Description
One, technical field
The invention belongs to biological technical field, relate to a kind of method of quick production hydrilla verticillata high grade project seedling.
Two, background technology
Hydrilla verticillata (Hydrilla verticillata Royle) is the black a kind of aquatic herbaceous plant of Trentepohlia of Hydrocharctaceae, is grown in the freshwater lake.This plant has very high tolerance to water pollution, can serve as water purification instrument kind and revegetation pioneer in eutrophication water restoration of the ecosystem engineering.This plant is also extensively planted in fish, shrimp and the crab pool, not only can also can provide necessary taking refuge and the spawning place for it for above-mentioned aquatic products provide bait.In addition, in indoor appreciation aquiculture process, this plant also be use always make the scape material.
In the market the hydrilla verticillata seedling is in great demand.But about the research of hydrilla verticillata all concentrate on its natural population distribution, its resistance to soiling and purifying water effect, with and application in fish shrimp crab is cultured.In practice process, the hydrilla verticillata provenance all is collected in natural environment.Root system suffers destruction to a certain degree more in the salvaging process, and not of uniform size again between the plant individuality, is unfavorable for that it successfully transplants.In addition, the hydrilla verticillata majority of natural source carries disease germs, and is unfavorable for the safe handling in the fish shrimp crab pool and the indoor appreciation aquarium.Mainly with the hibernaculum breeding, provenance is limited in the natural environment for last hydrilla verticillata, and excessive collection causes the destruction in its primary habitat easily.
Do not see the research and the report of the cultivation and the extensive artificial propagation thereof of relevant hydrilla verticillata aseptic seedling so far both at home and abroad.
Three, summary of the invention
The objective of the invention is: utilize plant tissue culture technique, obtain and at indoor large-scale breeding hydrilla verticillata engineering seedling, and keep it aseptic as far as possible, to overcome open-air limited and with serious pollution drawback, the needs of the freshwater lake revegetation of adaptation China, open-air fish shrimp crab aquaculture and indoor appreciation Shui nationality setting collected of these species.
Technical scheme of the present invention is:
1. the collection of the original provenance of hydrilla verticillata and preparation
Gather the hydrilla verticillata plant from open-air lake autumn and winter, the stem section of clip band hibernaculum is soaked in suds more than the 2h, after cleaning with running water, cultivates in the greenhouse.Change running water every day, and add an amount of Hoagland macro-element nutrients liquid (seeing Table 1) weekly.In the greenhouse about intensity of illumination 1000lx, illumination every day 10h, about 25 ℃ of temperature.
Table 1.Hoagland macro-element nutrients formula of liquid is:
| Reagent name | Consumption (every L) |
| Ca(NO 3) 2 | 0.821g |
| KNO 3 | 0.506g |
| MgSO 4·7H 2O | 0.616g |
| KH 2PO 4 | 0.272g |
| Fe-EDTA Na 2-EDTA FeSO 4·7H 2O | 7.45mg 5.57mg |
* remove the heating earlier of two kinds of compositions of Fe-EDTA in the mentioned reagent and boil together, all the other each compositions all each personal distilled water are made into 100 times of mother liquors, and the time spent mixes each composition with running water and prevents precipitation, and pH is adjusted into 6.0.
2. the acquisition of hydrilla verticillata aseptic seedling
Get the above-mentioned hibernaculum of in the greenhouse, cultivating more than 2 weeks, with 70% alcohol immersion certain hour, NaClO bleaching agent and a certain proportion of Tween-80 with debita spissitudo soaks certain hour again, after the thorough rinsing of sterile water, inoculate in the sub-1/2Ms liquid nutrient medium and (see Table 2), in group training chamber, cultivate.About intensity of illumination 1500lx, illumination every day 10h, temperature still is about 25 ℃.Note during this time observing, reject the material that still pollutes at any time.
3. the large-scale breeding of hydrilla verticillata aseptic seedling
After aseptic individual in the above-mentioned material was cultivated through 20d, most cauline leafs obviously extended, and plant is sturdy.Material is divided into the segment that saves with 2～3 in the ultra-clean operating room, is inoculated in the culture fluid of identical component.Each segment is after a period of time cultivates, and most meetings go out lateral bud the trifle director.Treat to downcut after lateral bud is grown up, can continue to cut apart, be inoculated in the 1/2Ms culture fluid, to obtain the greater amount stem eye; Also can directly lateral bud be downcut, be transferred in the 1/4Ms liquid nutrient medium, most roots that will grow 1～3cm length behind the 20d.Above-mentioned cultivation is also carried out illumination and temperature-resistant in group training chamber.
4. strong sprout of hydrilla verticillata engineering seedling
More than the long 6cm of stem in the 1/4Ms medium, the hydrilla verticillata of well developed root system takes out, and can directly satisfy the needs of indoor appreciation Shui nationality setting.Transplant if be used for large-size lake revegetation and the fish shrimp crab pool, must be transplanted to the bottom earlier and be covered with in the big glass jar of sandstone of the bacterium of going out.Keep changing water weekly and adding a certain amount of Hoagland nutrient solution more than the depth of water 50cm to promote its growth.When outdoor daytime, temperature was more than 20 ℃, glass jar is placed outdoor, directly utilize solar irradiation; Otherwise, still be put in the greenhouse, keep temperature and replenish artificial lighting.When growing to 40cm, plant can transplant on a large scale when above.
5. the application of hydrilla verticillata engineering seedling
Gather in the crops the above healthy hydrilla verticillata plant of the long 6cm of stem in the 1/4Ms culture fluid, after with running water culture fluid residual on the plant being rinsed well,, plant in the indoor appreciation aquarium according to the needs of setting.Gather in the crops the above healthy hydrilla verticillata plant of 40cm in the big glass jar, adopt proper density, be transplanted in the target lake separately or with other water biological species, be used for target ground revegetation and purification of water quality, or be transplanted in the fish shrimp crab pool, back also the providing for it as the fish shrimp crab feed of group is provided takes refuge and the spawning place.
Table 2.Ms liquid nutrient medium basic recipe is:
| Reagent name | Consumption (mg/L) | Reagent name | Consumption (mg/L) |
| Macroelement | Trace element | ||
| NH 4NO 3 | 1650 | KI | 0.83 |
| KNO 3 | 1900 | H 3BO 3 | 6.2 |
| CaCl 2·2H 2O | 440 | MnSO 4·4H 2O | 22.3 |
| MgSO 4·7H 2O | 370 | ZnSO 4·7H 2O | 10.6 |
| KH 2PO 4 | 170 | Na 2MoO 4·2H 2O | 0.25 |
| Organic principle | CuSO 4·5H 2O | 0.025 | |
| Inositol | 100 | CoCl 2·6H 2O | 0.025 |
| Nicotinic acid | 0.5 | The Fe-EDTA agent that boils together | |
| Puridoxine hydrochloride | 0.5 | Na2-EDTA | 37.3 |
| Thiamine hydrochloride | 0.1 | FeSO 4-7H 2O | 27.8 |
| Glycine | 2.0 | ||
| Sucrose | 30000 |
* above-mentioned composition except that sucrose is made into 4 kinds of 50 times of mother liquors by macroelement, trace element, organic principle and the Fe-EDTA agent that boils together with distilled water, the time spent, sucrose was existing molten with distilled water mixed diluting again.Adjust pH to 5.7 back branch and install in the 100mL triangular flask, with sealing film wrapping back in 121 ℃, 0.1MPa left and right sides moist heat sterilization 18min.
Effect of the present invention is:
1) obtained the hydrilla verticillata aseptic seedling.The hydrilla verticillata raw material of field acquisition itself are seriously polluted, and this pasture and water cauline leaf delicacy has absorbing capacity again concurrently, makes its sterilization process have suitable difficulty.Present technique adopts suds to embathe, and cultivates certain hour in advance in the nutrient solution of running water cooperation low concentration, preliminary earlier reduction pollution level, and raising material vigor.By ethanol and the acting in conjunction of NaClO bleaching agent, thoroughly sterilize again.Select the hibernaculum of hydrilla verticillata in the present technique for use, help the resistance of material itself disinfectant.By this program, guaranteeing that the material survival rate is also more than 50% on the basis of aseptic rate more than 60%.
2) set up hydrilla verticillata engineering seedling fast speed breeding approach.Present technique directly utilizes its lateral bud to cut apart breeding on the basis that obtains the hydrilla verticillata aseptic seedling, and step is simple, and is easy to operate, and agents useful for same is the chemical product that routinizes, and is with low cost, is convenient to its large-scale industrialized production.Be one with 20d and cut apart the cycle, each aseptic hibernaculum or lateral bud may be partitioned into 4 stem sections with 2～3 trifles at least, and 3 lateral buds and each stem section tillers at least estimate that with this each original bud can be told (4 * 3) in 1 year at least
17Individual sprouting.And each young shoot grows up to the above whole plant of 6cm and needs 40d approximately from being erupted into, estimate with this, one long * wide be 8 layers of common culturing rack of 120cm * 60cm, can be more than 25000 strains of year production hydrilla verticillata engineering seedling.
This invention can be regardless of season, views and admires setting for lake revegetation, the breed of the fish shrimp crab pool and the indoor Shui nationality at any time enough hydrilla verticillata high grade project seedlings are provided.
Four, embodiment
Example 1. is gathered the hydrilla verticillata provenance in Taihu Lake, east November from Suzhou, and the stem section of clip band hibernaculum is used the running water rinsing behind the immersion 4h in suds.Cultivate in the greenhouse with the 2L beaker.Change running water every day, and add the Hoagland nutrient solution weekly one time.In the greenhouse about intensity of illumination 1000lx, illumination every day 10h, about 25 ℃ of temperature.It is sturdy to choose cane behind the 15d, 30 pieces of the hibernaculums of blade N/D, with 70% alcohol immersion several seconds, soaked several minutes with dilution proper proportion and the NaClO bleaching agent that contains a certain amount of Tween-80 again, after the thorough rinsing of sterile water, be inoculated in and contain the sealing in the triangular flask of 40mL 1/2Ms liquid nutrient medium, in group training chamber, cultivate.Average intensity of illumination 1500lx, illumination every day 10h, about 25 ℃ of temperature.After this observe every day, reject contaminated materials at any time, get 21 pieces of aseptic buds after 1 week.Most hibernaculums grow to more than the 4cm behind the 20d, and have the part hibernaculum to grow lateral bud again at base portion.Under aseptic condition, downcut the long stem apex bud section of 1～2cm, be transferred in the 1/4Ms liquid nutrient medium.Remain stem section material in addition with per 2～3 brief summaries segmentation again, continue to cultivate in the 1/2Ms culture fluid.Again through 20d, in 1/4Ms, have 25 bud sections and develop into band and take root in strain.These whole plants are transferred to are used to view and admire Shui nationality setting after cultivating a period of time again in the 1/4 new culture fluid.The material of cultivating in 1/2Ms simultaneously also differentiates 92 sproutings through 20d, continues cutting and cultivates.
Example 2. is gone bail for and is had in the greenhouse hydrilla verticillata stem section of band hibernaculum, soaks in suds behind the 3h with the thorough rinsing of running water, cultivates in the running water that flows again.Adopt natural lighting, about 25 ℃ of temperature.It is sturdy to cut cane behind the 48h, 50 pieces of the hibernaculums of blade N/D, with 70% alcohol immersion several seconds, soaked several minutes with dilution proper proportion and the NaClO bleaching agent that contains a certain amount of Tween-80 again, after the thorough rinsing of sterile water, be inoculated in and contain the sealing in the triangular flask of 40mL 1/2Ms liquid nutrient medium, in group training chamber, cultivate.About intensity of illumination 1500lx, illumination every day 10h, temperature still is 25 ℃.After this reject the material that still pollutes at any time, get 34 pieces of aseptic buds after 1 week.Under aseptic condition, culture all is cut into behind the 20d at least with the stem section of 2～3 brief summaries, continues to cultivate in the 1/2Ms culture fluid.Through 20d, obtain 413 sproutings altogether again, the sprouting of the above length of 1.5cm is wherein taken off, be transferred in the 1/4Ms culture fluid, cultivate 30d again, obtain the healthy seedling of 356 strains altogether.These seedling replantings in the big glass jar of the sandstone that are covered with the bacterium of going out, are used for muskeg and recover research.
Claims (2)
1. a hydrilla verticillata expands numerous method, it is characterized in that utilizing the Plant Tissue Breeding principle, cultivate and sterilization process through pre-, obtain the aseptic hibernaculum of hydrilla verticillata, be incubated at after the segment in the 1/2Ms liquid nutrient medium to obtain more sproutings, again the bud section be transferred in the 1/4Ms liquid nutrient medium, continue to be cultured to more than the long 6cm of stem to induce the formation of root, forward in the big glass jar, replenish the Hoagland nutrient solution and continue strong sprout to more than the 40cm.
2. the application of hydrilla verticillata in the vegetation recovery engineering of lake that is obtained by the described method of claim 1 is characterized in that material length is to more than the 40cm.
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| CNB2004100138484A CN1317944C (en) | 2004-01-08 | 2004-01-08 | Wheel leaf black algae engineering seedling fast breeding method |
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| CNB2004100138484A CN1317944C (en) | 2004-01-08 | 2004-01-08 | Wheel leaf black algae engineering seedling fast breeding method |
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| CN1317944C true CN1317944C (en) | 2007-05-30 |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102124952A (en) * | 2011-01-21 | 2011-07-20 | 江苏九久环境科技有限公司 | Method for fast propagating hydrilla varticillata through tissue culture |
Families Citing this family (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102450213B (en) * | 2010-11-01 | 2013-04-17 | 南京中科水治理股份有限公司 | Method for improving propagation survival rate of hydrilla verticillata |
| CN103718956A (en) * | 2013-11-20 | 2014-04-16 | 青岛佰众化工技术有限公司 | Sterilization method for solieria explants |
| CN103651125A (en) * | 2013-11-22 | 2014-03-26 | 李本明 | Inducing method for callus of solieria |
| CN107333658A (en) * | 2017-09-13 | 2017-11-10 | 中冶华天工程技术有限公司 | A kind of quick-breeding method of the close thorn eel grass engineering seedling of hormone induction |
| CN108901849B (en) * | 2018-07-26 | 2022-03-08 | 水生藻安生物科技（武汉）有限公司 | Tissue culture method of aseptic seedlings of hydrilla verticillata |
| CN109220769B (en) * | 2018-11-15 | 2020-09-22 | 中国水产科学研究院黄海水产研究所 | Method for improving commodity yield and product quality of caulerpa lentillifera |
| CN109287490B (en) * | 2018-11-29 | 2021-07-30 | 南京大学 | Method for rapidly propagating high-quality seedling of aquatic plant water soft-shelled turtle and application |
| CN111543327A (en) * | 2020-06-11 | 2020-08-18 | 南大（常熟）研究院有限公司 | Production method of detoxified seedlings suitable for rapid propagation of submerged plants |
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Non-Patent Citations (1)
| Title |
|---|
| 蛋白质新资源——黑藻的研究 文明 盛哲 林亲众，湖南农学院学报，第20卷第5期 1994 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102124952A (en) * | 2011-01-21 | 2011-07-20 | 江苏九久环境科技有限公司 | Method for fast propagating hydrilla varticillata through tissue culture |
| CN102124952B (en) * | 2011-01-21 | 2012-07-25 | 江苏九久环境科技有限公司 | Method for fast propagating hydrilla varticillata through tissue culture |
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| CN1555682A (en) | 2004-12-22 |
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