CN103053422A - Method for reproducing potamogeton perfoliatus - Google Patents
Method for reproducing potamogeton perfoliatus Download PDFInfo
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- CN103053422A CN103053422A CN2013100072861A CN201310007286A CN103053422A CN 103053422 A CN103053422 A CN 103053422A CN 2013100072861 A CN2013100072861 A CN 2013100072861A CN 201310007286 A CN201310007286 A CN 201310007286A CN 103053422 A CN103053422 A CN 103053422A
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Abstract
The invention discloses a method for reproducing potamogeton perfoliatus, belonging to the field of biotechnology. The method comprises the following steps of: collecting potamogeton perfoliatus, removing leaves and roots, washing for 1-2 hours with water, taking stems with 2-3 nodes at top ends, and washing the stems with distilled water, disinfecting and sterilizing the stems with ethanol and sodium hypochlorite sequentially; inoculating the stems in a culture bottle filled with a culture medium, culturing for 30-35 days and then reproducing new plants, and sterilizing the culture medium at a high temperature. According to the method disclosed by the invention, 4-9 new plants can be reproduced by every 2-3 stems, and a non-infection rate achieves more than 95%; the culture period is 45 days and is short; and the survival rate of the new plants used as engineering seedlings after being domesticated is greater than 99%, the technical problems of insufficient quantity of potamogeton perfoliatus seedlings, low survival rate, high quantity of pollutants and the like are solved, and potamogeton perfoliatus which is sterile and high in survival rate can be rapidly and massively reproduced.
Description
Technical field
The invention belongs to biological technical field, be specifically related to the propagation method that a kind of submerged plant is worn the leaf pondweed.
Background technology
Lake, Reservoir Eutrophication problem is day by day serious in recent years, and aquatic ecosystem is subject to heavy damage.Submerged plant is one of primary producer in the aquatic ecosystem, has purifying water and slows down the ability that bed mud discharges, and by the allelopathy of algae, suppresses the Growth and reproduction of algae.Wear the function that the leaf pondweed is applied to have in the Aquatic Ecosystem 2 systems engineering Xi to be increased the submerged plant population diversity, improve aquatic ecosystem stability assurance water quality.Wearing the leaf pondweed is one of biomaterial main during Ecology makes up, repairs, regulates and control as higher aquatic plants.
Wearing the leaf pondweed is perennial heavy pasture and water basis, has flourishing rhizome, can both survive in the many water bodys of China, and water environment is had wider tolerance and stronger adaptive capacity.
Wear at present leaf pondweed seedling and mainly from natural river, lake, gather, owing to contain a large amount of pollutants from the seedling of natural world collection, such as algae, insect pest and larva ovum etc.When being applied to the Aquatic Ecosystem 2 systems engineering Xi, its plant gives birth to a little less than branch, the root of hair ability, and survival rate is low.Therefore have without dirt, take root and germinating capacity is strong wears the key that leaf pondweed seedling is successfully field planting by artificial culture.
Summary of the invention
The objective of the invention is to overcome Ecology makes up, repairs, regulates and control in the engineering, wear leaf pondweed seedling lazy weight, survival rate is low, the shortcomings such as pollutant is many, therefore the invention provides a kind of utilization wears the propagation method that leaf pondweed stem eye carries out a large amount of engineering seedlings of aseptic culture, the method can effectively solve and wear that the leaf pondweed is used for that Ecology makes up, repairs, during the regulation and control engineering, its seedling lazy weight, the shortcoming such as of poor quality, survival rate is low.
In order to solve the problems of the technologies described above, the present invention takes following technical scheme:
The method of leaf pondweed is worn in a kind of breeding, it is characterized in that comprising the steps:
1) the leaf pondweed is worn in collection, removes Ye Hegen, behind water flushing 1～2h, gets the stem that the top contains 2～3 joints, cleans stem with distilled water;
2) successively use ethanol and clorox to the stem sterilization;
3) stem is inoculated into cultivate in the blake bottle that medium is housed after 30～35 days and can breeds new plant, described medium is through high-temperature sterilization.
Further technical scheme is: the female parent that the new plant that cultivation is grown is cultivated as a new generation, repeat above-mentioned step 3), but wear leaf pondweed continuous culture many generations.
Further technical scheme is: place 1/2 Hoagland solution to practice seedling after 15 days new plant, can be used as the engineering seedling and use.
Further technical scheme is: the component that comprises following weight in every liter of the described medium: sucrose 20～30 grams, NAA0.1～0.3 milligram, 6～BA0.3～0.6 milligram, 1650 milligrams in ammonium nitrate, 1900 milligrams in potassium nitrate, 440 milligrams of calcium chloride dihydrates, 370 milligrams in magnesium sulfate, 170 milligrams of potassium dihydrogen phosphates, 0.83 milligram of potassium iodide, 6.2 milligrams of boric acid, 22.3 milligrams of manganese sulphates, 8.6 milligrams in zinc sulphate, 0.25 milligram of sodium molybdate, 0.025 milligram in copper sulphate, 0.025 milligram of cobalt chloride, 27.8 milligrams in ferrous sulfate, 37.3 milligrams of disodium ethylene diamine tetraacetates, 100 milligrams of inositols, 0.5 milligram in nicotinic acid, 0.5 milligram of puridoxine hydrochloride, 0.1 milligram of thiamine hydrochloride, 2 milligrams of glycine.
Further technical scheme is: the per 2 liters of components that comprise following weight of described 1/2 Hoagland solution: 945 milligrams in nitrate of lime, 607 milligrams in potassium nitrate, 115 milligrams in ammonium phosphate, 493 milligrams in magnesium sulfate, 13.9 milligrams in ferrous sulfate, 37.3 milligrams of disodium ethylene diamine tetraacetates, 0.83 milligram of potassium iodide, 6.2 milligrams of boric acid, 22.3 milligrams of manganese sulphates, 8.6 milligrams in zinc sulphate, 0.25 milligram of sodium molybdate, 0.025 milligram in copper sulphate, 0.025 milligram of cobalt chloride.
Further technical scheme is: the stem sterilization be may further comprise the steps with ethanol and clorox step 2): be that 50%～70% alcohol immersion was sterilized for 30～60 seconds with volume fraction, then wash 3 times with sterile distilled water; Be that 0.5～1% clorox soaks sterilization 10～20 minutes with mass fraction again, again with sterile distilled water flushing 3 times.
Further technical scheme is: the high-temperature sterilization described in the step 3) is under 120～125 degrees centigrade, sterilization 30～40min.
Further technical scheme is: the condition of cultivating step 3) is medium pH 5.8～6.0,20～28 ℃ of temperature, and intensity of illumination 1800～2500Lx, light application time is 8-12h every day.
The invention has the beneficial effects as follows:
1) utilize the stem of wearing the leaf pondweed to adopt aseptic culture medium to carry out aseptic culture, every 2-3 section stem can be bred the new plant of 4-9, reaches more than 95% without infection rate; The of future generation new plant that the new plant of adopting cultivation to grow is cultivated as female parent, reach more than 99% without infection rate, it is more than 99% that new plant is practiced the survival rate of using as the engineering seedling behind the seedling, has solved and has worn leaf pondweed seedling lazy weight, the technical problems such as survival rate is low, and pollutant is many.
2) adopting the present invention to cultivate the cycle of wearing the new plant of leaf pondweed is that cultivation cycle is short about 45 days, but the leaf pondweed is worn in the breeding of rapid, high volume, solves problems such as wearing leaf pondweed seedling lazy weight.
Required nutrition composition when the medium of 3) preparing among the present invention can satisfy the stem breeding of wearing the leaf pondweed promotes to wear the stem Fast-propagation of leaf pondweed.
Embodiment
Below in conjunction with specific embodiment of the present invention the present invention is made further explanation and description.
Embodiment 1:
The present embodiment, is bred altogether approximately 60000 strains by the aseptic culture in continuous 5 generations and is worn the leaf pondweed through the time of some months for to wear the leaf pondweed from field acquisition 20 strains.
The present embodiment may further comprise the steps:
Step 1: what gather robust growth in the natural water wears 20 strains of leaf pondweed, removes Ye Hegen, and water flushing 1-2h gets the top and contains 2-3 stem that saves, and with distilled water stem is being rinsed well.
Step 2: successively use ethanol and clorox to the stem sterilization: be 30～60 seconds of 50%～70% alcohol immersion sterilization with volume fraction first, then with sterile distilled water flushing 3 times; Be that 0.5～1% clorox soaks sterilization 10～20 minutes with mass fraction again, again with sterile distilled water flushing 3 times.
Step 3: preparation medium and medium high-temperature sterilization: the component that comprises following weight in every liter of medium: sucrose 20～30 grams, NAA0.1～0.3 milligram, 6～BA0.3～0.6 milligram, 1650 milligrams in ammonium nitrate, 1900 milligrams in potassium nitrate, 440 milligrams of calcium chloride dihydrates, 370 milligrams in magnesium sulfate, 170 milligrams of potassium dihydrogen phosphates, 0.83 milligram of potassium iodide, 6.2 milligrams of boric acid, 22.3 milligrams of manganese sulphates, 8.6 milligrams in zinc sulphate, 0.25 milligram of sodium molybdate, 0.025 milligram in copper sulphate, 0.025 milligram of cobalt chloride, 27.8 milligrams in ferrous sulfate, 37.3 milligrams of disodium ethylene diamine tetraacetates, 100 milligrams of inositols, 0.5 milligram in nicotinic acid, 0.5 milligram of puridoxine hydrochloride, 0.1 milligram of thiamine hydrochloride, 2 milligrams of glycine, all the other are distilled water.Medium pH is 5.8-6.0, and the medium high-temperature sterilization is under temperature 120-125 ℃, sterilization 30-40min.
Step 4: stem is inoculated in the medium cultivates: be that 75% alcohol sprays workbench with volume fraction before the inoculation, and with ultraviolet lamp to operation room and the workbench 20-30min that sterilizes.Stem behind step 2 sterilization is inoculated in the blake bottle that medium is housed, according to one embodiment of present invention, the medium that 100ml is housed in the blake bottle of every 250ml can be placed 2-3 section stem, then place 20～28 ℃ of temperature, intensity of illumination 1800～2500Lx, light application time is to cultivate under the condition of culture of 8-12h every day, cultivate after 30-35 days, the new plant of 4-9 strain is arranged in each blake bottle, and every strain is long to be 5-10cm, and pollution-free rate is more than 95%.
Step 5: the female parent with the new plant of step 4 cultivation is cultivated as a new generation, new plant is cut into the 2-3 section, repeat above-mentioned steps 4.
The pollution-free rate of the new plant in the per generation after two generations, the three generations who adopts new plant to cultivate reaches is more than 99%.
The present embodiment is through the some months time, in 5 generations of continuous culture, breeds altogether approximately 60000 strains and newly wears leaf pondweed plant.
Embodiment 2:
The present embodiment is for gathering 1000 strains of wearing leaf pondweed seedling, and through about 60 days, continuous culture can provide 20000 bottles of approximately 100000 strain new talents in per 45 days after two generations, was colonizated in the Ecology engineering water body, and survival rate can reach more than 99%.
The present embodiment comprises the steps:
Step 1: what gather robust growth wears 1000 strains of leaf pondweed, removes Ye Hegen, and water flushing 1-2h gets the top and contains 2-3 stem that saves, and with distilled water stem is being rinsed well.
Step 2: successively use ethanol and clorox to the stem sterilization: first with volume fraction being 30～60 seconds of 50%～70% alcohol immersion sterilization, then with sterile distilled water flushing 3 times; Be that 0.5～1% clorox soaks sterilization 10～20 minutes with mass fraction again, again with sterile distilled water flushing 3 times.
Step 3: preparation medium and medium high-temperature sterilization: the component that comprises following weight in every liter of medium: sucrose 20～30 grams, NAA0.1～0.3 milligram, 6～BA0.3～0.6 milligram, 1650 milligrams in ammonium nitrate, 1900 milligrams in potassium nitrate, 440 milligrams of calcium chloride dihydrates, 370 milligrams in magnesium sulfate, 170 milligrams of potassium dihydrogen phosphates, 0.83 milligram of potassium iodide, 6.2 milligrams of boric acid, 22.3 milligrams of manganese sulphates, 8.6 milligrams in zinc sulphate, 0.25 milligram of sodium molybdate, 0.025 milligram in copper sulphate, 0.025 milligram of cobalt chloride, 27.8 milligrams in ferrous sulfate, 37.3 milligrams of disodium ethylene diamine tetraacetates, 100 milligrams of inositols, 0.5 milligram in nicotinic acid, 0.5 milligram of puridoxine hydrochloride, 0.1 milligram of thiamine hydrochloride, 2 milligrams of glycine, all the other are distilled water.Medium pH is 5.8-6.0, and the medium high-temperature sterilization is under temperature 120-125 ℃, sterilization 30-40min.
Step 4: stem is inoculated in the medium cultivates: be that 75% alcohol sprays workbench with volume fraction before the inoculation, and with ultraviolet lamp to operation room and the workbench 20-30min that sterilizes.Stem behind step 2 sterilization is inoculated in the blake bottle that medium is housed, according to one embodiment of present invention, place the 100ml medium in the blake bottle of every 250ml and can place 2-3 section stem, then place 20～28 ℃ of temperature, intensity of illumination 1800～2500Lx, light application time is to cultivate under the condition of culture of 8-12h every day, cultivate after 30-35 days, the new plant of 4-9 strain is arranged in each blake bottle, and every strain is long to be 5-10cm, and pollution-free rate is more than 95%.
Step 5: new plant is cut into the 2-3 section, carries out two cultures as the female parent of two cultures, inoculate altogether 5000 bottles, repeat above-mentioned steps 4, cultivate and obtain new plant of two generations, pollution-free rate is more than 99%.
Step 6: the new plant of two generations that step 5 is cultivated carries out three cultures as the female parent of three cultures, and new plant is cut into the 2-3 section, inoculates altogether 25000 bottles, repeats above-mentioned steps 4, obtains the new plant of three generations.
Step 7: 5000 bottles new plant in the new plant of three generations is cultivated as follow-on maternal the continuation, repeated above-mentioned steps 4.
Step 8: prepare 1/2 Hoagland solution: per 2 liter of 1/2 Hoagland solution comprises the component of following weight: 945 milligrams in nitrate of lime, 607 milligrams in potassium nitrate, 115 milligrams in ammonium phosphate, 493 milligrams in magnesium sulfate, 13.9 milligrams in ferrous sulfate, 37.3 milligrams of disodium ethylene diamine tetraacetates, 0.83 milligram of potassium iodide, 6.2 milligrams of boric acid, 22.3 milligrams of manganese sulphates, 8.6 milligrams in zinc sulphate, 0.25 milligram of sodium molybdate, 0.025 milligram in copper sulphate, 0.025 milligram of cobalt chloride, remaining is distilled water.
Step 9: place 1/2 Hoagland solution to practice seedling after 15 days new plant remaining 20000 bottles in the new plant of three generations, namely can be used for the engineering seedling and use, practice seedling and can improve plant to the adaptability of natural environment, improve survival rate etc.
Utilize 5000 bottles plant as maternal continuous culture, can provide 100000 strains to wear leaf pondweed engineering seedling in average per 45 days, survival rate can reach more than 99%.
Although invention has been described with reference to explanatory embodiment of the present invention here, above-described embodiment only is the better embodiment of the present invention, embodiments of the present invention are not restricted to the described embodiments, should be appreciated that, those skilled in the art can design a lot of other modification and embodiments, and these are revised and embodiment will drop within the disclosed principle scope and spirit of the application.
Claims (8)
1. the method for leaf pondweed is worn in a breeding, it is characterized in that comprising the steps:
1) the leaf pondweed is worn in collection, removes Ye Hegen, behind water flushing 1～2h, gets the stem that the top contains 2～3 joints, cleans stem with distilled water;
2) successively use ethanol and clorox to the stem sterilization;
3) stem is inoculated into cultivate in the blake bottle that medium is housed after 30～35 days and can breeds new plant, described medium is through high-temperature sterilization.
2. the method for leaf pondweed is worn in breeding according to claim 1, characterized by further comprising following step: the female parent with new plant is cultivated as a new generation, repeat above-mentioned steps 3).
3. the method for leaf pondweed is worn in breeding according to claim 1 and 2, characterized by further comprising following step: place 1/2 Hoagland solution hardening after 15 days new plant, use as the engineering seedling.
4. the method for leaf pondweed is worn in breeding according to claim 1, it is characterized in that comprising in every liter of the described medium component of following weight: sucrose 20～30 grams, NAA0.1～0.3 milligram, 6～BA0.3～0.6 milligram, 1650 milligrams in ammonium nitrate, 1900 milligrams in potassium nitrate, 440 milligrams of calcium chloride dihydrates, 370 milligrams in magnesium sulfate, 170 milligrams of potassium dihydrogen phosphates, 0.83 milligram of potassium iodide, 6.2 milligrams of boric acid, 22.3 milligrams of manganese sulphates, 8.6 milligrams in zinc sulphate, 0.25 milligram of sodium molybdate, 0.025 milligram in copper sulphate, 0.025 milligram of cobalt chloride, 27.8 milligrams in ferrous sulfate, 37.3 milligrams of disodium ethylene diamine tetraacetates, 100 milligrams of inositols, 0.5 milligram in nicotinic acid, 0.5 milligram of puridoxine hydrochloride, 0.1 milligram of thiamine hydrochloride, 2 milligrams of glycine.
5. the method for leaf pondweed is worn in breeding according to claim 3, it is characterized in that the per 2 liters of components that comprise following weight of described 1/2 Hoagland solution: 945 milligrams in nitrate of lime, 607 milligrams in potassium nitrate, 115 milligrams in ammonium phosphate, 493 milligrams in magnesium sulfate, 13.9 milligrams in ferrous sulfate, 37.3 milligrams of disodium ethylene diamine tetraacetates, 0.83 milligram of potassium iodide, 6.2 milligrams of boric acid, 22.3 milligrams of manganese sulphates, 8.6 milligrams in zinc sulphate, 0.25 milligram of sodium molybdate, 0.025 milligram in copper sulphate, 0.025 milligram of cobalt chloride.
6. the method for leaf pondweed is worn in breeding according to claim 1, it is characterized in that step 2) described in ethanol and clorox the stem sterilization be may further comprise the steps: be 30～60 seconds of 50%～70% alcohol immersion sterilization with volume fraction, then with sterile distilled water flushing 3 times; Be that 0.5～1% clorox soaks sterilization 10～20 minutes with mass fraction again, again with sterile distilled water flushing 3 times.
7. the method for leaf pondweed is worn in breeding according to claim 1, it is characterized in that the high-temperature sterilization described in the step 3) is under 120～125 degrees centigrade, sterilization 30～40min.
8. the method for leaf pondweed is worn in breeding according to claim 1, it is characterized in that step 3) described in the condition of cultivating be medium pH 5.8～6.0,20～28 ℃ of temperature, intensity of illumination 1800～2500Lx, light application time is 8-12h every day.
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Cited By (2)
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| CN104705194A (en) * | 2015-04-03 | 2015-06-17 | 南京大学 | Hormone-induced rapid propagation method of potamogeton pectinatus engineering seedlings |
| CN106954549A (en) * | 2017-02-27 | 2017-07-18 | 水生藻安生物科技（武汉）有限公司 | A kind of comb tooth bog pondweed tissue culture and rapid proliferation method |
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Cited By (3)
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| CN104705194A (en) * | 2015-04-03 | 2015-06-17 | 南京大学 | Hormone-induced rapid propagation method of potamogeton pectinatus engineering seedlings |
| CN106954549A (en) * | 2017-02-27 | 2017-07-18 | 水生藻安生物科技（武汉）有限公司 | A kind of comb tooth bog pondweed tissue culture and rapid proliferation method |
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| CN103053422B (en) | 2014-03-26 |
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Application publication date: 20130424 Assignee: Chengdu Wide Horizon New Town Development Co., Ltd. Assignor: Chengdu Huadu Agricultural Development Co., Ltd. Contract record no.: 2017510000017 Denomination of invention: Method for reproducing potamogeton perfoliatus Granted publication date: 20140326 License type: Exclusive License Record date: 20170626 |
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Application publication date: 20130424 Assignee: Chengdu Qingyuan Water Environment Treatment Co., Ltd Assignor: Chengdu Huadu Agricultural Development Co., Ltd. Contract record no.: X2019510000007 Denomination of invention: Method for reproducing potamogeton perfoliatus Granted publication date: 20140326 License type: Common License Record date: 20191204 Application publication date: 20130424 Assignee: Hainan Wanhua Investment Co., Ltd Assignor: Chengdu Huadu Agricultural Development Co., Ltd. Contract record no.: X2019510000005 Denomination of invention: Method for reproducing potamogeton perfoliatus Granted publication date: 20140326 License type: Common License Record date: 20191203 |
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