CN110915655A - Treatment method for promoting germination of curculigo orchioides seeds - Google Patents
Treatment method for promoting germination of curculigo orchioides seeds Download PDFInfo
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- CN110915655A CN110915655A CN201911211232.0A CN201911211232A CN110915655A CN 110915655 A CN110915655 A CN 110915655A CN 201911211232 A CN201911211232 A CN 201911211232A CN 110915655 A CN110915655 A CN 110915655A
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
- A01H4/00—Plant reproduction by tissue culture techniques ; Tissue culture techniques therefor
- A01H4/005—Methods for micropropagation; Vegetative plant propagation using cell or tissue culture techniques
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
- A01H4/00—Plant reproduction by tissue culture techniques ; Tissue culture techniques therefor
- A01H4/001—Culture apparatus for tissue culture
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Abstract
The invention discloses a treatment method for promoting germination of curculigo orchioides seeds, which comprises the steps of collecting mature curculigo orchioides seeds, removing seed coats, soaking in gibberellin for 24 hours, and carrying out aseptic germination, wherein the concentration of the gibberellin is 100 mg/L; sterile germination is to sterilize the gibberellin-treated seeds on a superclean bench, and inoculate the sterilized seeds to a culture medium for culture under proper culture conditions; sterilizing the seeds with 75% ethanol for 30s, washing with sterile water for 2 times, sterilizing with 0.1% mercuric chloride for 8min, and washing with sterile water for 5 times; the suitable culture conditions are 25 ℃, 2500Lx and 12 h/d; the composition of the culture medium is 1.5mg/L of MS +6-BA +0.8mg/L, and the pH value is 5.8. The method combines a unique method which is not found by others and is beneficial to promoting the germination of the seeds of the curculigo orchioides with a traditional seed germination method, so that the germination rate of the seeds of the curculigo orchioides reaches more than 83 percent.
Description
Technical Field
The invention relates to the technical field of seed biology and the technical field of medicinal plant biology, in particular to a method which comprises the following steps: a germination accelerating method and a seed sterile culture method which adopt a specific treatment mode to break seed coat barriers of curculigo orchioides seeds so as to improve the seed germination rate.
Background
The rhizoma Curculiginis is dried rhizome of Curculigo orchioides Gaertn. Harvesting perennial herbs in autumn and winter, removing root heads and fibrous roots, cleaning, and drying. Curculigo orchioides is a scarce medicinal plant, and has the effects of tonifying kidney yang, strengthening bones and muscles and dispelling cold-dampness. Can be used for treating sexual impotence, cold essence, flaccidity of tendons and bones, cold pain of waist and knees, and cold diarrhea due to yang deficiency. The wild resources of the curculigo orchioides are rich, but the destruction is serious and the reduction is rapid due to the large amount of digging. Curculigo orchioides grows mainly in forests, grasslands and barren slopes at an elevation below 1600 m. The curculigo orchioides are distributed in clusters, the distribution range is narrow, the quantity is small, the natural updating capability is poor, the fruiting quantity is extremely small, the plant growth vigor is weak, and the capability of resisting natural disasters is poor. The population is less young plants, the plant age is larger, the whole population presents a decline type, and the population is basically in an endangered state.
The curculigo orchioides is mainly propagated through seeds and roots, the seed setting rate of the curculigo orchioides is low, the seed propagation is difficult to realize, and the artificial cultivation is generally achieved through digging wild resources as seed sources and propagating through roots and roots. The curculigo orchioides seeds have dormancy characteristics and hardly germinate naturally under normal conditions.
The seed coat structure affects the air and water permeability of the seed, and some seed coats also have a mechanical barrier to seed embryo growth. The seeds of the curculigo orchioides are long round or oval, the seed coats are black, the surfaces are keratinized and hard; the surface of the seed has wrinkles or warty bulges, and the hilum, the pearl hole, the ridge and the joint are all obviously protruded.
The inventor invents a method for germinating the seeds and finds a culture medium suitable for the rapid germination of the seeds by comprehensively analyzing the mechanism for inhibiting the germination of the seeds of the curculigo orchioides and combining the characteristics of the germination of the seeds. No relevant report about the germination of the seeds of the curculigo orchioides is found at home and abroad through the search from 2017 to 2019.
Disclosure of Invention
The invention aims to solve the technical problem that the Curculigo orchioides seeds are difficult to germinate and have long germination time in the prior art, thereby providing a new method which is different from other methods and can greatly improve the germination rate (up to 83%) of the Curculigo orchioides seeds.
The technical scheme of the invention is as follows:
a treatment method for promoting germination of Curculigo orchioides seeds comprises collecting mature Curculigo orchioides seeds, removing seed coats, soaking in gibberellin, and performing aseptic germination, and is characterized in that collected mature Curculigo orchioides fruit pods or cracked seeds are dried in the shade, then the seeds are taken out of the fruit pods, the seed coats are removed, the gibberellin soaking treatment is performed, and then the seeds are germinated under aseptic conditions;
soaking the gibberellin for 24 hours, wherein the concentration of the gibberellin is 100 mg/L;
the sterile germination is to sterilize the seeds treated by gibberellin on a superclean bench, and inoculate the sterilized seeds to a culture medium for culture under proper culture conditions;
the sterilization treatment comprises sterilizing the seeds with 75% ethanol for 30s, washing with sterile water for 2 times, sterilizing with 0.1% mercuric chloride for 8min, and washing with sterile water for 5 times;
the suitable culture condition is carried out at 25 ℃ and 2500Lx for 12 h/d;
the composition of the culture medium is MS +6-BA1.5mg/L +0.8mg/L, and the pH value is 5.8.
The seed coat removing step is that the seeds dried in the shade are placed in a mortar, covered by gauze and slightly ground and crushed, and the seeds with the seed coats removed are picked out.
The invention has the beneficial effects that:
1. and adopting a measure of removing the testa from the seeds. The seeds of the curculigo orchioides have low germination rate and long germination time in a natural state, and the conventional production method of the curculigo orchioides seedlings is realized by rhizome propagation; the method removes the episperm before sowing seeds, aims to break the seed coat barrier of the curculigo orchioides seeds, improves the water absorption efficiency and the germination of embryos, has breakthrough progress in the aspect of seed germination, and improves the seed germination rate by 60 percent under the measure.
2. Adopting gibberellin soaking measures. The seeds without the testa are soaked in 100mg/L gibberellin solution for 24h, so that the testa can be swelled, the permeability of the testa is increased, and foreign substances can enter the testa; on the other hand, the soaking of gibberellin is high in the aspect of improving the germination rate of seeds, and the germination rate of the seeds is improved by 1.67%.
3. And adopting an orthogonal experimental design measure. The three factors of the basic culture medium type, the cytokinin concentration and the auxin concentration are considered, the culture medium which is most suitable for the germination of the curculigo orchioides seeds is determined, the seed germination rate is greatly improved, and the seed germination rate reaches 83.33%.
4. The seed peeling sterile germination method adopted by the invention simultaneously ensures that the seeds are not rotten in the germination process, and overcomes the problem that the seeds are rotten firstly due to long germination time in the traditional germination process.
5. The instrument used in the invention is simple and convenient to operate, so that the industrial production of the seedlings of the curculigo orchioides is easy to carry out.
Detailed Description
The present invention will be described in detail with reference to examples.
Example 1: sterile culture of curculigo orchioides seeds:
the main apparatus is as follows: a sterilizing pot and an ultra-clean workbench.
The curculigo orchioides seeds of example 1 were aseptically cultured according to the following procedure:
(1) picking seeds: picking off the ripe seed pods, and drying the seeds with the peels in the shade; and (4) dropping part of the burst seeds into soil, and collecting the seeds for later use by using tweezers.
(2) Seed screening: rubbing open the fruit pod dried in the shade, removing impurities, placing the seeds and the seeds scattered in the soil into water for screening, removing the floating seeds on the upper layer, collecting the robust and plump seeds on the lower layer, cleaning, drying in the shade, and storing in a refrigerator at 4 ℃ for later use.
(3) Preparing explant cleaning fluid: 3 drops of liquid detergent and 1 drop of 84 disinfectant are added into 1L of distilled water, the mixture is stirred evenly, and seed cleaning liquid is reserved.
(4) Cleaning explants: soaking the seeds in the solution for 3min, stirring, cleaning, draining after 1min, washing with distilled water for several times, draining, and placing into sterile bottle.
(5) Preparing a culture medium: the composition of the culture medium is MS +6-BA1.0mg/L + NAA0.5mg/L, and simultaneously, 30g/L of cane sugar, 7g/L of agar and 0.3mg/L of active carbon are added.
In this example and all examples of the present invention, the MS medium used in the preparation of the medium for sterile culture was a formulation commonly used in the art.
The preparation of MS culture medium is carried out according to the following table, firstly, 20 times mother liquor of macroelements is prepared, and all organic elements, ferric salt and trace elements are 100 times liquor. Taking 50mL of macroelement mother liquor, 10mL of organic element mother liquor, 10mL of ferric salt mother liquor and 10mL of trace element mother liquor for every 1L of MS culture medium, simultaneously adding 30g of cane sugar into every 1L of MS culture medium, and adjusting the pH value to 5.8.
TABLE 1 MS culture Medium formulation
Murashige and Skoog,1962
(6) Subpackaging and sterilizing a culture medium: after the preparation of the culture medium is finished, the culture medium is subpackaged by using tissue culture bottles, about 30mL of the culture medium is poured into each bottle, the bottles are covered and placed into a sterilization pot, and the sterilization is carried out for 20min at the temperature of 121 ℃ and under the pressure of 1.2 kpa. And taking out after sterilization, and standing for later use after cooling.
(7) And (3) explant sterilization: placing the sterile bottle filled with the seeds, the empty sterile bottle, sterile water, 75% alcohol, 0.1% mercuric chloride solution and tween 80 into a clean bench. Spraying alcohol on both hands, placing on a clean bench, opening sterile bottle containing seeds, pouring 20mL of 75% alcohol, pouring out alcohol rapidly after 30s, pouring sterile water, and washing twice for 1min each time. Pouring 20mL of 0.1% mercuric chloride solution, adding 1 drop of Tween 80, shaking to make it uniformly contact with seeds, pouring out the liquid after 8min, and washing with sterile water for 5 times, each time for 1 min. Draining water for later use.
(8) Inoculation: taking out sterilized rhizoma Curculiginis seeds, inoculating the seeds into culture medium with tweezers, and covering with bottle cap, wherein the number of seeds per bottle is 5.
(9) Culturing: placing the inoculated culture medium in a culture chamber, wherein the culture conditions are 25 +/-2 ℃, the light intensity is 2000Lx, and the illumination time is 12 h/d.
Example 2: germination comparison of intact seeds and seeds without testa:
example 2 is to examine the effect of seed coat removal on germination of seeds of Curculigo orchioides by comparing intact seeds with those from which the episperm has been removed.
The method for removing the episperm of the curculigo orchioides seeds comprises the following steps:
(1) the curculigo orchioides seeds screened in example 1 were taken out, about 50 seeds were placed in a mortar each time, a piece of gauze 15 × 15cm was placed over the mortar, and the gauze was ground with a mortar stick with a force controlled to break the seed coat without damaging the inside of the seeds.
(2) The peeled seeds were picked with small-size sharp tweezers and placed in 5mL centrifuge tubes, 60 seeds per tube.
(3) And (4) continuously grinding the unpeeled seeds until all the seeds in the mortar are completely peeled, and discarding the seed coats after the seeds are completely peeled.
The seeds of Curculigo orchioides without testa were used as experimental group and the unpeeled seeds were used as control group, and aseptically cultured for germination according to the method of example 1. Each group treated 60 seeds, representing 3 replicates. And recording the germination vigor, the germination rate and the germination condition of the seeds in the germination process.
TABLE 2 complete seed and Seed episperm seed Germination results
The experimental results show that: within 1 month, the germination rate of the curculigo orchioides seeds without the testa removal (control group) was 0, and the germination rate of the curculigo orchioides seeds without the testa removal was 60%.
Example 3: gibberellins (GA)3) The influence of the soaking concentration on the germination of the seeds of the curculigo orchioides:
example 3 an optimal treatment concentration was selected by soaking gibberellins at different concentrations and comparing the effect of different gibberellin concentrations on germination of seeds of Curculigo orchioides.
Placing the seeds of rhizoma Curculiginis with testa removed into the container at concentrations of 10mg/L, 50mg/L, 100mg/L, and 150mg/L respectively200mg/L and 300mg/L of GA3Soaking in the solution for 24h, and performing sterile germination of seeds by using distilled water as a control. The procedure was followed to carry out example 1.
TABLE 3 Effect of infusion treatment with gibberellins at different concentrations on Curculigo orchioides seed Germination
The experimental results show that: when the concentration of gibberellin is 50mg/L, the germination rate of the curculigo orchioides seeds is 61.67%, so that the optimal concentration of the curculigo orchioides seeds for soaking treatment is 50 mg/L.
Example 4: screening the optimal culture medium for sterile germination of the curculigo orchioides seeds:
through the investigation of three factors of basic medium type, cytokinin (6-BA) and auxin (NAA), orthogonal experiments are designed, and a culture medium suitable for the rapid germination of the seeds of the curculigo orchioides is screened out.
The seeds of curculigo orchioides without testa are taken as the material, and aseptic germination is carried out according to the formula in the following table and the method of example 1.
TABLE 4 factor level table
TABLE 5L9(34) Orthogonal experimental design table
TABLE 6 Effect of different types of media on Curculigo orchioides seed Germination
As can be seen from the experimental results in Table 6, the germination rate of Curculigo orchioides seeds in the culture medium of treatment 3, that is, in the culture medium of MS +6-BA1.5mg/L + NAA0.8mg/L, can be as high as 83.33%.
On the basis of deeply researching the physiological characteristics of the curculigo orchioides seeds and summarizing the research results of seed germination of other species, the inventor compares a unique method which is not found by others and is beneficial to promoting the seed germination with a traditional method which is not subjected to the treatment, so that the seeds germinate rapidly, the seed germination rate is improved to 83.33%, and an unexpected technical effect is generated. The instrument used in the invention is simple and convenient to operate, so that the industrial production of the seedlings of the curculigo orchioides is easy to carry out.
Finally, it is noted that the above-mentioned embodiments illustrate rather than limit the invention, and that, although the above-mentioned examples have described the process of the invention in detail, it will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention as defined by the appended claims.
Claims (2)
1. A treatment method for promoting germination of Curculigo orchioides seeds comprises the steps of collecting mature Curculigo orchioides seeds, removing seed coats, soaking in gibberellin, and aseptically germinating, and is characterized in that: drying collected mature curculigo orchioides fruit pods or the seeds which are burst out in the shade, taking the seeds out of the fruit pods, removing seed coats and soaking the seeds in gibberellin, and then germinating the seeds under the aseptic condition;
soaking the gibberellin for 24 hours, wherein the concentration of the gibberellin is 100 mg/L;
the sterile germination is to sterilize the seeds treated by gibberellin on a superclean bench, and inoculate the sterilized seeds to a culture medium for culture under proper culture conditions;
the sterilization treatment comprises sterilizing the seeds with 75% ethanol for 30s, washing with sterile water for 2 times, sterilizing with 0.1% mercuric chloride for 8min, and washing with sterile water for 5 times;
the suitable culture condition is carried out at 25 ℃ and 2500Lx for 12 h/d;
the composition of the culture medium is 1.5mg/L of MS +6-BA +0.8mg/L, and the pH value is 5.8.
2. The treatment method for promoting germination of seeds of curculigo orchioides as claimed in claim 1, wherein: the seed coat removing step is that the seeds dried in the shade are placed in a mortar, covered by gauze and slightly ground and crushed, and the seeds with the seed coats removed are picked out.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN117567201A (en) * | 2024-01-17 | 2024-02-20 | 云南省农业科学院药用植物研究所 | Method for improving domestication survival rate and growth rate of seedling of Xian Mao Zupei |
| CN117598065A (en) * | 2024-01-19 | 2024-02-27 | 云南省农业科学院药用植物研究所 | Method for promoting quick germination of curculigo seeds |
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| CN107027624A (en) * | 2017-04-28 | 2017-08-11 | 云南农业大学 | A kind of method for obtaining polygerm Yunnan Paris polyphylla seedling |
| CN108157074A (en) * | 2018-01-04 | 2018-06-15 | 云南农业大学 | A kind of implantation methods that thizoma curculiginis is promoted to yield positive results |
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| CN107027624A (en) * | 2017-04-28 | 2017-08-11 | 云南农业大学 | A kind of method for obtaining polygerm Yunnan Paris polyphylla seedling |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN117567201A (en) * | 2024-01-17 | 2024-02-20 | 云南省农业科学院药用植物研究所 | Method for improving domestication survival rate and growth rate of seedling of Xian Mao Zupei |
| CN117567201B (en) * | 2024-01-17 | 2024-03-15 | 云南省农业科学院药用植物研究所 | Method for improving domestication survival rate and growth rate of seedling of Xian Mao Zupei |
| CN117598065A (en) * | 2024-01-19 | 2024-02-27 | 云南省农业科学院药用植物研究所 | Method for promoting quick germination of curculigo seeds |
| CN117598065B (en) * | 2024-01-19 | 2024-03-26 | 云南省农业科学院药用植物研究所 | Method for promoting quick germination of curculigo seeds |
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