CN107318303B - Method for promoting paris polyphylla seeds to rapidly germinate into seedlings - Google Patents
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- CN107318303B CN107318303B CN201710692082.4A CN201710692082A CN107318303B CN 107318303 B CN107318303 B CN 107318303B CN 201710692082 A CN201710692082 A CN 201710692082A CN 107318303 B CN107318303 B CN 107318303B
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- 241000244987 Daiswa polyphylla Species 0.000 title claims abstract description 57
- 238000000034 method Methods 0.000 title claims abstract description 40
- 230000001737 promoting effect Effects 0.000 title claims abstract description 18
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 49
- 238000007654 immersion Methods 0.000 claims abstract description 40
- XEYBHCRIKKKOSS-UHFFFAOYSA-N disodium;azanylidyneoxidanium;iron(2+);pentacyanide Chemical compound [Na+].[Na+].[Fe+2].N#[C-].N#[C-].N#[C-].N#[C-].N#[C-].[O+]#N XEYBHCRIKKKOSS-UHFFFAOYSA-N 0.000 claims abstract description 29
- 229940083618 sodium nitroprusside Drugs 0.000 claims abstract description 29
- 230000001954 sterilising effect Effects 0.000 claims abstract description 26
- 239000012153 distilled water Substances 0.000 claims abstract description 12
- 238000002791 soaking Methods 0.000 claims abstract description 12
- 238000012258 culturing Methods 0.000 claims abstract description 6
- 238000011109 contamination Methods 0.000 claims abstract description 3
- 230000035784 germination Effects 0.000 claims description 44
- 238000004659 sterilization and disinfection Methods 0.000 claims description 16
- 239000008223 sterile water Substances 0.000 claims description 12
- 238000004140 cleaning Methods 0.000 claims description 9
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 7
- 238000005406 washing Methods 0.000 claims description 6
- 229920000742 Cotton Polymers 0.000 claims description 4
- 238000001035 drying Methods 0.000 claims description 4
- 241000894006 Bacteria Species 0.000 claims description 3
- 229960000318 kanamycin Drugs 0.000 claims description 3
- 229930027917 kanamycin Natural products 0.000 claims description 3
- SBUJHOSQTJFQJX-NOAMYHISSA-N kanamycin Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CN)O[C@@H]1O[C@H]1[C@H](O)[C@@H](O[C@@H]2[C@@H]([C@@H](N)[C@H](O)[C@@H](CO)O2)O)[C@H](N)C[C@@H]1N SBUJHOSQTJFQJX-NOAMYHISSA-N 0.000 claims description 3
- 229930182823 kanamycin A Natural products 0.000 claims description 3
- LWJROJCJINYWOX-UHFFFAOYSA-L mercury dichloride Chemical compound Cl[Hg]Cl LWJROJCJINYWOX-UHFFFAOYSA-L 0.000 claims description 3
- 229960003255 natamycin Drugs 0.000 claims description 3
- NCXMLFZGDNKEPB-FFPOYIOWSA-N natamycin Chemical compound O[C@H]1[C@@H](N)[C@H](O)[C@@H](C)O[C@H]1O[C@H]1/C=C/C=C/C=C/C=C/C[C@@H](C)OC(=O)/C=C/[C@H]2O[C@@H]2C[C@H](O)C[C@](O)(C[C@H](O)[C@H]2C(O)=O)O[C@H]2C1 NCXMLFZGDNKEPB-FFPOYIOWSA-N 0.000 claims description 3
- 235000010298 natamycin Nutrition 0.000 claims description 3
- 239000004311 natamycin Substances 0.000 claims description 3
- 239000000853 adhesive Substances 0.000 claims description 2
- 230000001070 adhesive effect Effects 0.000 claims description 2
- 239000002390 adhesive tape Substances 0.000 claims description 2
- 238000001816 cooling Methods 0.000 claims description 2
- 230000000249 desinfective effect Effects 0.000 claims description 2
- 230000015572 biosynthetic process Effects 0.000 claims 1
- 230000000694 effects Effects 0.000 description 7
- 229930191978 Gibberellin Natural products 0.000 description 6
- IXORZMNAPKEEDV-UHFFFAOYSA-N gibberellic acid GA3 Natural products OC(=O)C1C2(C3)CC(=C)C3(O)CCC2C2(C=CC3O)C1C3(C)C(=O)O2 IXORZMNAPKEEDV-UHFFFAOYSA-N 0.000 description 6
- 239000003448 gibberellin Substances 0.000 description 6
- 238000004519 manufacturing process Methods 0.000 description 6
- 241001251761 Riparia Species 0.000 description 5
- 239000000203 mixture Substances 0.000 description 5
- MWUXSHHQAYIFBG-UHFFFAOYSA-N Nitric oxide Chemical compound O=[N] MWUXSHHQAYIFBG-UHFFFAOYSA-N 0.000 description 4
- 238000007789 sealing Methods 0.000 description 4
- 230000008901 benefit Effects 0.000 description 3
- 238000009395 breeding Methods 0.000 description 3
- 230000001488 breeding effect Effects 0.000 description 3
- 230000007547 defect Effects 0.000 description 3
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- 230000007226 seed germination Effects 0.000 description 3
- 231100000331 toxic Toxicity 0.000 description 3
- 230000002588 toxic effect Effects 0.000 description 3
- 241000196324 Embryophyta Species 0.000 description 2
- 238000005520 cutting process Methods 0.000 description 2
- 239000003814 drug Substances 0.000 description 2
- 238000005516 engineering process Methods 0.000 description 2
- 239000000463 material Substances 0.000 description 2
- 230000014284 seed dormancy process Effects 0.000 description 2
- 238000013517 stratification Methods 0.000 description 2
- 238000009423 ventilation Methods 0.000 description 2
- XFXPMWWXUTWYJX-UHFFFAOYSA-N Cyanide Chemical compound N#[C-] XFXPMWWXUTWYJX-UHFFFAOYSA-N 0.000 description 1
- -1 GA are used3CTK Substances 0.000 description 1
- 206010061218 Inflammation Diseases 0.000 description 1
- 241000234280 Liliaceae Species 0.000 description 1
- 206010028980 Neoplasm Diseases 0.000 description 1
- 241000416918 Polyphylla Species 0.000 description 1
- 241000283984 Rodentia Species 0.000 description 1
- 239000003242 anti bacterial agent Substances 0.000 description 1
- 229940088710 antibiotic agent Drugs 0.000 description 1
- 208000006673 asthma Diseases 0.000 description 1
- 230000000740 bleeding effect Effects 0.000 description 1
- 239000013043 chemical agent Substances 0.000 description 1
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- 230000003467 diminishing effect Effects 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 230000007614 genetic variation Effects 0.000 description 1
- 239000005556 hormone Substances 0.000 description 1
- 229940088597 hormone Drugs 0.000 description 1
- 230000004054 inflammatory process Effects 0.000 description 1
- 230000002401 inhibitory effect Effects 0.000 description 1
- 238000011081 inoculation Methods 0.000 description 1
- 239000007788 liquid Substances 0.000 description 1
- 229960002523 mercuric chloride Drugs 0.000 description 1
- 239000002840 nitric oxide donor Substances 0.000 description 1
- 210000000056 organ Anatomy 0.000 description 1
- 239000003375 plant hormone Substances 0.000 description 1
- 238000012545 processing Methods 0.000 description 1
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- 238000007873 sieving Methods 0.000 description 1
- 230000011664 signaling Effects 0.000 description 1
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Classifications
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01C—PLANTING; SOWING; FERTILISING
- A01C1/00—Apparatus, or methods of use thereof, for testing or treating seed, roots, or the like, prior to sowing or planting
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01C—PLANTING; SOWING; FERTILISING
- A01C1/00—Apparatus, or methods of use thereof, for testing or treating seed, roots, or the like, prior to sowing or planting
- A01C1/02—Germinating apparatus; Determining germination capacity of seeds or the like
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01C—PLANTING; SOWING; FERTILISING
- A01C1/00—Apparatus, or methods of use thereof, for testing or treating seed, roots, or the like, prior to sowing or planting
- A01C1/08—Immunising seed
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- Life Sciences & Earth Sciences (AREA)
- Soil Sciences (AREA)
- Environmental Sciences (AREA)
- Health & Medical Sciences (AREA)
- Physiology (AREA)
- Pretreatment Of Seeds And Plants (AREA)
Abstract
The invention provides a method for promoting paris polyphylla seeds to germinate into seedlings quickly, which comprises the following steps: pretreating picked paris polyphylla seeds; checking the tightness of the intermittent immersion type bioreactor, and then sterilizing the intermittent immersion type bioreactor; soaking pretreated rhizoma paridis seeds with water overnight to make the seeds fully swell and become mellow, sterilizing the seeds under aseptic condition, and confirming that the seeds are sterilized thoroughly without contamination; culturing and germinating: and (3) placing the seeds completely sterilized in the step into an intermittent immersion bioreactor for culture, adding sterilized distilled water containing SNP into the intermittent immersion bioreactor, inoculating the paris polyphylla seeds, and culturing in a dark environment, wherein the intermittent immersion frequency of the seeds is 3-5 minutes/6 h. The method of combining sodium nitroprusside and the intermittent immersion type bioreactor can lead the paris polyphylla seeds to germinate rapidly.
Description
Technical Field
The invention relates to a method for rapidly germinating paris polyphylla seeds into seedlings by combining Sodium Nitroprusside (SNP) with an intermittent immersion bioreactor, belonging to the technical field of rare medicinal plant propagation.
Background
Paris is a perennial herb of Paris of Liliaceae, and is an important Chinese medicinal material, wherein the dried rhizome tissues of Paris polyphylla and Paris polyphylla are identified as the medicine source of Paris polyphylla which is a commonly used medicinal material in Chinese pharmacopoeia (2015 edition). Modern pharmacology indicates that the rhizome of the paris polyphylla has the effects of tranquilizing and easing pain, relieving asthma, stopping bleeding, inhibiting bacteria and diminishing inflammation and the like, and particularly has obvious curative effect on tumor resistance in recent years. Therefore, under the drive of economic benefit, the wild resources of the paris polyphylla are continuously dug by plunder, and are endangered to be extinct. Therefore, the wild resources of the paris polyphylla can be protected by artificial propagation, and the ever-increasing market demand can be met.
The current common artificial propagation method in production is mainly rhizome propagation method. The rhizome cutting propagation is the most successful at present, but has the defects and shortcomings. First, the rhizome is an economic organ, which increases the cost after cutting, and the amount of the used root is large. Second, it requires a lot of seeds, is subject to decay, and is subject to rodent attack. Thirdly, the propagation coefficient is high, but the period is long, and the production requirement cannot be met. The artificial breeding by utilizing the seeds has the advantages of large propagation coefficient, low production cost and the like, can avoid the consumption of the medicinal part by the propagation of the rhizome, greatly improve the propagation coefficient, reduce the genetic variation risk caused by tissue culture and is an effective way for relieving the shortage of paris polyphylla resources. However, the source of the paris polyphylla is severely limited by the defect that the paris polyphylla grows slowly, seeds are sowed in a natural state and need to germinate in two winter and one summer, and then the seeds continue to grow for 5 to 7 years before being used as medicines. Therefore, artificial breaking of seed dormancy and improvement of germination rate become important subjects for large-scale artificial breeding of Paris polyphylla.
At present, the temperature swing stratification is used for promoting the germination of the paris polyphylla seeds by adopting a plurality of methods, namely, the seeds are alternately treated at high temperature and low temperature, so that the seeds are promoted to germinate in advance, but the treatment time in the process is longer, and generally 120-200 days are needed. In addition, plant hormones such as GA are used3CTK, chemical agent PEG and the like can also promote the germination of seeds to different degrees by treating the paris polyphylla seeds. Although these seed breeding techniques can promote seed germination to some extent, it has been found in actual production that stratification and hormone treatment are still time consuming. Moreover, due to the fact that the germination period of the paris polyphylla seeds is long, manual water replenishing, air exchange and other work are often needed in the germination promoting process, and therefore rotting and mildewing of the seeds caused by improper control of water and air conditions often occur, a more efficient and rapid method needs to be researched to promote the germination of the seeds, and the situation that paris polyphylla is scarce is fundamentally solved.
Nitric oxide is a gaseous signaling molecule that has the effect of breaking plant seed dormancy. Sodium Nitroprusside (SNP) is a nitric oxide donor, the water solution of the SNP is capable of promoting the germination of seeds, and the early research in the laboratory finds that the seeds of the paris polyphylla can be rapidly promoted to germinate by soaking the seeds of the paris polyphylla with the sodium nitroprusside water solution with proper concentration. However, the sodium nitroprusside solution can release toxic cyanide while providing nitric oxide, so that the long-time soaking treatment can have toxic effect on seeds. On the contrary, if the soaking time is too short, the effect of effectively promoting germination cannot be obtained.
Disclosure of Invention
In order to overcome the defects of troublesome seed treatment operation and long germination time in the existing paris polyphylla seed germination technology, the invention provides a method for rapidly germinating paris polyphylla seeds by combining sodium nitroprusside with a batch immersion bioreactor.
The technical scheme adopted for realizing the above purpose of the invention is as follows:
a method for promoting paris polyphylla seeds to rapidly germinate into seedlings comprises the following steps: (1) pretreating picked paris polyphylla seeds: removing testa from picked mature rhizoma paridis seeds, washing, drying the seeds in the shade indoors, and then placing at 0-4 ℃ for later use;
(2) checking the tightness of the intermittent immersion type bioreactor, and then sterilizing the intermittent immersion type bioreactor;
(3) soaking the paris polyphylla seeds obtained in the step (1) with water overnight to ensure that the seeds are fully imbibed and plump, then sterilizing the seeds under the aseptic condition, and confirming that the sterilization is thorough and has no contamination phenomenon;
(4) culturing and germinating: placing the seeds completely sterilized in the previous step into intermittent immersion bioreactors for culture, adding sterilized distilled water into the intermittent immersion bioreactors, wherein each liter of sterilized distilled water contains 50-150 mu mol of SNP, inoculating 350-500 grains of paris polyphylla seeds into each intermittent immersion bioreactor, the immersion frequency of the paris polyphylla seeds in the intermittent immersion bioreactors is 3-5 minutes/6 hours, carrying out culture in a dark environment, recording the initial time of seed radicle breakthrough in the culture process, and periodically counting the germination rate of the seeds and the percentage of radicle length of the seeds which is more than or equal to 3 cm.
In the process of removing the paris polyphylla testa in the step (1), the surface of the seed cannot be damaged, and after the testa is removed, the paris polyphylla seed is washed by clean water for 2-5 times, so that the residue of the testa is completely removed; meanwhile, after the seeds are washed clean, the seeds are not exposed to the sun and dried, so that the germination rate of the seeds is not influenced.
The method for checking the tightness in the step (2) is that 1L clear water is filled in the intermittent immersion type bioreactor, the tightness of the intermittent immersion type bioreactor is observed, the intermittent immersion type bioreactor added with the clear water is connected to a pump, and whether the filter tip flows backwards or not is observed.
The sterilization method in the step (2) is as follows: putting the cleaned intermittent immersion bioreactor into an autoclave for sterilization, wrapping the bioreactor with paper after cooling, performing secondary sterilization, wrapping the upper layer of the bioreactor with the paper, locking the upper layer with cotton threads, wrapping the base with the paper, continuously locking the base with a second cotton thread, sticking the sharp part with transparent adhesive, sticking the base with adhesive tape, and preventing the sharp object from puncturing the base after sterilization.
The method for sterilizing the seeds in the step (3) is as follows: cleaning seeds with sterile water for 3-5 times in an ultra-clean workbench, then disinfecting with 75% alcohol by volume concentration for 30-45 s, immediately cleaning with sterile water for 3-5 times after disinfection, soaking with 0.1-0.2% mercury bichloride by mass concentration for 10-15 min, and cleaning with sterile water for 5 times;
sterilizing the culture dish, paving 2 layers of filter paper at the bottom of the culture dish after sterilization, and dripping sterile water into the culture dish until the filter paper is just wetted; and (3) respectively filling the sterilized seeds into culture dishes for culture, independently culturing for 5 days in small portions, observing whether the seeds are sterilized completely, and then transferring the seeds which are not infected with bacteria into an intermittent immersion bioreactor.
In the step (4), when the seeds which are completely sterilized are transferred into the intermittent immersion type bioreactor, the operation is carried out on a superclean bench, and after the periphery of the cover of the intermittent immersion type bioreactor is sterilized by an alcohol lamp, the cover of the reactor can be opened.
In step (4), 1L sterilized distilled water was added to each batch immersion bioreactor, and each liter of the sterilized distilled water contained 150mg kanamycin and 50mg natamycin.
Compared with the existing germination promoting and seedling raising technology of the paris polyphylla, the intermittent immersion method provided by the invention not only can achieve the purpose of interval immersion, but also can realize automatic ventilation, so that the germination promoting effect of SNP can be fully exerted, the toxic effect on seeds is not generated, and the work of manually supplementing moisture to the seeds, ventilating and the like is not needed in the germination promoting period. The method is simple to operate, saves labor and effectively reduces the probability of seed rotting and mildewing caused by improper control of moisture and ventilation conditions. In addition, the method greatly shortens the germination time and improves the germination rate. Experiments prove that the germination rate of the invention can reach more than 90 percent in 75 days, the strong seedling rate of the germinated seeds (the ratio of the seeds with radicle length more than or equal to 3cm after germination-greenhouse and field experiments show that the seeds with radicle length more than or equal to 3cm are easy to survive) is also obviously improved, and the production cost is obviously reduced. The method ensures that the germination rate and the strong seedling rate of the paris polyphylla seeds reach the optimal combination state, and is more economical and effective.
Detailed Description
The present invention will be described in detail with reference to specific examples, but the scope of the present invention is not limited to the examples.
Example 1
(1) Mature seeds of paris polyphylla picked in the current year are removed of glume shells, then the seeds are placed into a breathable gauze bag, red testa is rubbed off by hands, and then the paris polyphylla seeds are washed for 3-4 times by flowing clear water, so that the testa residues are thoroughly removed. Then placing the mixture on newspaper, drying the mixture indoors in the shade for 3-5 days, and placing the mixture in a refrigerator at 4 ℃ for later use.
(2) Washing the novel reactor with clean water, then filling 1L clean water into the reactor, observing the sealing property of the reactor, connecting the reactor with the clean water to a pump, observing whether the filter tip flows backwards, placing the sealed reactor into an autoclave for sterilization at 121 ℃, carrying out 20 minutes, wrapping the reactor with newspaper after the reactor is cooled, and carrying out sterilization again.
(3) Soaking the seeds of Paris polyphylla which are dried in the shade in clear water for 24 hours to ensure that the seeds are fully imbibed and become mellow. Then, in a superclean workbench, cleaning the seeds for 3-5 times by using sterile water; then using 75% alcohol to disinfect for 30s, and washing with sterile water for 3-5 times; soaking the mixture in 0.1% mercuric chloride solution for 10-15 min, and washing the mixture with sterile water for 5 times; finally, the culture is carried out in a 150mm culture dish paved with 2 layers of filter paper (the culture dish is sterilized in advance), sterile water is dripped to just wet the filter paper, and the culture dish is sealed by a sealing film. And (3) placing the seeds into an artificial climate incubator for dark culture at 25 +/-2 ℃, observing the seeds after 5 days of culture, if mould grows on the filter paper, cleaning the mould seeds and the culture dish in the culture dish, and transferring the seeds which are thoroughly sterilized into a reactor for culture.
(4) Sterilizing the periphery of the cover of the reactor by using an alcohol burner, opening the cover of the reactor, then pouring sterile distilled water (1L) containing 50-150 mu mol/L SNP or 500 mg/L GA3l into the reactor, adding antibiotics (150 mg/L kanamycin and 50 mg/L natamycin) into each treatment, inoculating 400 grains of Paris polyphylla seeds into each reactor, after the inoculation is finished, screwing down the screws, and sealing a sealing film at gaps.
(5) The reactor was placed in a dark place, the frequency of reactor immersion was 3 minutes/6 hours, the culture conditions were 25. + -. 1 ℃ temperature, 60% relative humidity, and the culture time was 105 days. Recording the time of the first-seen radicle of the seed and counting the germination rate every other time day, wherein the mark of the seed germination takes the condition that the radicle breaks through the seed coat just as the standard, and the strong seedling rate takes the proportion of the seeds with the radicle length more than or equal to 3cm as the standard.
In this example, the optimum concentration of GA verified in the early stage of the laboratory was used in combination with clean water3(500 mg/L) as a control, sodium nitroprusside solutions with different concentrations are arranged to soak seeds, and the effect of using the 100 mu mol/L sodium nitroprusside solution is the best, the seed coat is broken through at 35 days, the time is 3 days and 9 days before gibberellin and clear water respectively, and at 75 days, the 100 mu mol/L sodium nitroprusside solution is dissolvedThe germination rate of the seeds in the liquid treatment can reach 90 percent, is higher than that of gibberellin treatment, and is far higher than that of other concentrations and clear water treatment, at the 105 th day, the strong seedling rate reaches 73.2 percent, and is obviously better than that of a control and 500 mg/L GA3And processing shows that the 100 mu mol/L sodium nitroprusside solution is used for soaking the seeds to obviously promote the early germination of the paris polyphylla seeds and obviously improve the strong seedling rate of the germinated seeds.
Example 2
Selecting Yunnan rhizoma paridis seeds picked in the current year for germination experiment, removing glume, manually rubbing off red episperm with hands, slowly sieving off episperm with a fine-hole sieve, washing Paris polyphylla seeds with flowing clear water for 3-4 times, and completely removing episperm residues. Then placing on newspaper, drying in the shade for 3-5 days indoors, and placing in a refrigerator at 4 deg.C for use.
Soaking the seeds of Paris polyphylla dried in the shade in clear water for 24h to ensure that the seeds are fully imbibed and become mellow. And then sterilizing the seeds in a superclean workbench, putting the seeds into an artificial climate incubator for dark culture at 20 +/-2 ℃, observing the seeds after 5 days of culture, if mould grows on the filter paper, cleaning the mould seeds and the culture dish in the culture dish, and transferring the thoroughly sterilized seeds into a reactor for culture.
After the cover of the reactor is sterilized by an alcohol burner, the cover of the reactor is opened, 1L sterilized distilled water containing 100 mu mol/L SNP is added, 1L sterilized distilled water is used as a control, 3 times of repeated tests are carried out, 100 seeds are added each time, and the result is that 20.33 percent of the radicle of the seeds breaks through the seed coat at 45 days, the germination rate of the paris polyphylla seeds reaches 91.01 percent at 75 days, and the seedling strengthening rate reaches 75.4 percent at 105 days in 100 mu mol/L SNP treatment.
According to the method, through laboratory germination statistics, the applicant finds that seeds cultured by the method have the best effect when the 100 mu mol/L sodium nitroprusside solution is used, break through seed coats in 35 days, are 3 days and 9 days earlier than gibberellin and clear water respectively, the germination rate of the seeds in the treatment of the 100 mu mol/L sodium nitroprusside solution can be up to 90 percent and is higher than that of the seeds treated by gibberellin and the treatment of the clear water in 75 days, the seeds are soaked by the 100 mu mol/L sodium nitroprusside solution to obviously promote the conventional early germination of the paris polyphylla seeds and improve the germination rate of the paris polyphylla seeds, the sodium nitroprusside solution has a better effect and is more economical and practical in price than that of the gibberellin treatment, the sodium nitroprusside solution has the advantages in production, the capacity of an intermittent bioreactor is 270mm, the diameter is 270mm, the lower volume of the seeds is 1.5, the lower volume of the sodium nitroprusside solution is higher than that of the gibberellin treatment, the germination rate of the sodium nitroprusside solution is higher than that of the sodium nitroprusside solution, the sodium nitroprusside solution is higher than that the sodium nitroprusside solution, the germination rate of the conventional germination promotion of the germinate group is higher than that the germination of the riparian, the germination of the riparia polyphylla strain is increased, the germination of the riparia napestra riparia napestrus strain in 75, the conventional method, the germination promotion of the germination of the riparia napestrus strain is increased by using the germination promotion of the riparia napestrus strain.
Claims (7)
1. A method for promoting paris polyphylla seeds to rapidly germinate into seedlings is characterized by comprising the following steps: (1) pretreating picked paris polyphylla seeds: removing testa from picked mature rhizoma paridis seeds, washing, drying the seeds in the shade indoors, and then placing at 0-4 ℃ for later use;
(2) checking the tightness of the intermittent immersion type bioreactor, and then sterilizing the intermittent immersion type bioreactor;
(3) soaking the paris polyphylla seeds obtained in the step (1) with water overnight to ensure that the seeds are fully imbibed and plump, then sterilizing the seeds under the aseptic condition, and confirming that the sterilization is thorough and has no contamination phenomenon;
(4) culturing and germinating: placing the seeds completely sterilized in the previous step into intermittent immersion bioreactors for culture, adding sterilized distilled water into the intermittent immersion bioreactors, wherein each liter of sterilized distilled water contains 100 mu mol of sodium nitroprusside, inoculating 350-500 grains of paris polyphylla seeds into each intermittent immersion bioreactor, the immersion frequency of the paris polyphylla seeds in the intermittent immersion bioreactors is 3-5 minutes/6 hours, carrying out culture in a dark environment, recording the initial time of seed radicle breakthrough in the culture process, and periodically counting the germination rate of the seeds and the percentage of radicle length of the seeds which is more than or equal to 3 cm.
2. The method for promoting the rapid germination of the paris polyphylla seeds into seedlings according to claim 1, wherein the method comprises the following steps: in the process of removing the paris polyphylla testa in the step (1), the surface of the seed cannot be damaged, and after the testa is removed, the paris polyphylla seed is washed for 2-5 times by using clear water, so that the residue of the testa is completely removed; meanwhile, after the seeds are washed clean, the seeds are not exposed to the sun and dried, so that the germination rate of the seeds is not influenced.
3. The method for promoting the rapid germination and seedling formation of paris polyphylla seeds of claim 1, wherein the tightness checking in step (2) is performed by filling 1L clear water into the batch immersion bioreactor, observing the tightness, connecting the batch immersion bioreactor into which the clear water is added to a pump, and observing whether the filter tip flows backwards.
4. The method for promoting the rapid germination of the paris polyphylla seeds into seedlings according to claim 1, wherein the method comprises the following steps: the sterilization method in the step (2) is as follows: putting the cleaned intermittent immersion bioreactor into an autoclave for sterilization, wrapping the bioreactor with paper after cooling, performing secondary sterilization, wrapping the upper layer of the bioreactor with the paper, locking the upper layer with cotton threads, wrapping the base with the paper, continuously locking the base with a second cotton thread, sticking the sharp part with transparent adhesive, sticking the base with adhesive tape, and preventing the sharp object from puncturing the base after sterilization.
5. The method for promoting the rapid germination of the paris polyphylla seeds into seedlings according to claim 1, wherein the method comprises the following steps: the method for sterilizing the seeds in the step (3) is as follows: cleaning seeds with sterile water for 3-5 times in an ultra-clean workbench, then disinfecting with 75% alcohol by volume concentration for 30-45 s, immediately cleaning with sterile water for 3-5 times after disinfection, soaking with 0.1-0.2% mercury bichloride by mass concentration for 10-15 min, and cleaning with sterile water for 5 times;
sterilizing the culture dish, paving 2 layers of filter paper at the bottom of the culture dish after sterilization, and dripping sterile water into the culture dish until the filter paper is just wetted; and (3) respectively filling the sterilized seeds into culture dishes for culture, independently culturing for 5 days in small portions, observing whether the seeds are sterilized completely, and then transferring the seeds which are not infected with bacteria into an intermittent immersion bioreactor.
6. The method for promoting the rapid germination of the paris polyphylla seeds into seedlings according to claim 1, wherein the method comprises the following steps: in the step (4), when the seeds which are completely sterilized are transferred into the intermittent immersion type bioreactor, the operation is carried out on a superclean bench, and after the periphery of the cover of the intermittent immersion type bioreactor is sterilized by an alcohol lamp, the cover of the reactor can be opened.
7. The method for promoting the rapid germination of Paris polyphylla seeds into seedlings according to claim 1, wherein in the step (4), 1L sterilized distilled water is added into each batch immersion type bioreactor, and each liter of the sterilized distilled water contains 150mg of kanamycin and 50mg of natamycin.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710692082.4A CN107318303B (en) | 2017-08-14 | 2017-08-14 | Method for promoting paris polyphylla seeds to rapidly germinate into seedlings |
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| CN105210647B (en) * | 2015-10-28 | 2017-12-26 | 云南祥仁生物科技有限公司 | A kind of method for massively culturing of Yunnan Paris polyphylla seedling |
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