CN111771451A - Method for determining vitality of michelia sphaerana seeds - Google Patents
Method for determining vitality of michelia sphaerana seeds Download PDFInfo
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- CN111771451A CN111771451A CN202010710310.8A CN202010710310A CN111771451A CN 111771451 A CN111771451 A CN 111771451A CN 202010710310 A CN202010710310 A CN 202010710310A CN 111771451 A CN111771451 A CN 111771451A
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- 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
- A01C1/025—Testing seeds for determining their viability or germination capacity
Abstract
The invention provides a method for measuring the viability of michelia sphaerana seeds, which belongs to the field of measurement of the germination capacity of seeds and comprises the steps of manually cleaning red aril of the seeds to obtain pure seeds; carrying out pre-wetting treatment on the pure seeds to obtain pre-wetted seeds; removing endocarp from the prewetted seeds, and cutting off 2-6 mm endosperm on two sides of the seeds respectively to obtain seeds to be dyed; and (3) placing the seeds to be dyed in a tetrazolium dyeing solution for dyeing treatment to obtain dyed seeds, wherein the dyeing is carried out under the dark condition at the temperature of 28-32 ℃ for 20-72 h, and when the embryos are uniformly dyed into red and the endosperms are dyed into red, the vitality of the seeds is indicated. The method fills the blank of the method for determining the vitality of the michelia globosa seeds, defines the TTC dyeing method of the michelia globosa seeds and the judgment of different conditions after dyeing, and provides reference for quickly and effectively detecting the vitality of the michelia globosa.
Description
Technical Field
The invention relates to the technical field of determination of seed germination capacity, in particular to a method for determining the vitality of michelia sphaerana seeds.
Background
Michelia globosa is an evergreen arbor of Michelia of Magnoliaceae, is a national secondary protective plant, and is listed as a local tree species in the national precious tree species cultivation base construction engineering project. The sphagna smilax has beautiful tree appearance, pleasant flower fragrance, and various characteristics of leaves and polymerized fruits, and is a landscaping tree species with high ornamental value. The michelia sphaeracea has the characteristics of high growth speed, adaptability, drought resistance, cold resistance, dust absorption, strong pollution resistance and the like, and can be used as an important evergreen, aromatic and environment-friendly high-quality greening tree species. With the increasingly prominent role of michelia globosa in the greening industry, people pay more attention to the utilization and new variety cultivation. However, the michelia sphaeroida seed coat is hard, has a limitation effect on moisture absorption and respiratory metabolism, is a low-degree recalcitrant seed with moderate complex morphological physiological dormancy, has the characteristic of deep dormancy, has a low seed germination rate, and has important significance for guiding sowing production and the like because of the polymorphism of the seed, different seed dormancy degrees and different germination.
Seed viability refers to the potential ability of a seed to germinate or the viability of an embryo. The viability of the seeds is an important index of seed quality, and has important significance in crop breeding, seed production, seed processing and other works. Tetrazole (TTC) determination is the most important method for determining seed viability widely used at present, but the TTC method has many problems in actual operation, and firstly, due to reasons such as specific method selection or improper operation of some key links, the determination value is far lower than the true value, for example, in the TTC method, if the dyeing time and the seed treatment time are not well mastered, the determination result is lower than the true value; secondly, a range is given in certain key steps, for example, the time for soaking and dyeing is 3-8 h, which may be different for specific species. Therefore, in the determination of the seed viability, for a specific species, the selection of the determination method, the key steps and the like should be changed accordingly according to the characteristics of a specific seed, so as to form a specific and effective method. Research finds that the TTC determination technology is relatively focused on grain crops such as rice, wheat, corn and the like, but no clear operation flow and judgment standard exist in the tetrazole dyeing method for the michelia sphaera seeds at present.
Disclosure of Invention
In view of this, the invention aims to provide a method for determining the viability of michelia sphaerantha seeds, and the method provided by the invention can be used for quickly and accurately detecting the viability of michelia sphaerantha seeds.
In order to achieve the above object, the present invention provides the following technical solutions:
the invention provides a method for measuring the viability of michelia sphaerana seeds, which comprises the following steps:
1) manually cleaning red aril of the seeds to obtain pure seeds;
2) carrying out pre-wetting treatment on the pure seeds to obtain pre-wetted seeds;
3) removing endocarp from the prewetted seeds, and cutting off 2-6 mm endosperm on two sides of the seeds respectively to obtain seeds to be dyed;
4) placing the seeds to be dyed in a tetrazolium dyeing solution for dyeing treatment to obtain dyed seeds, wherein the dyeing is carried out under a dark condition, the dyeing temperature is 28-32 ℃, and the dyeing time is 20-72 hours;
5) the dyed seeds were dissected along the embryo, and when the embryo was evenly dyed red and the endosperm was dyed red, it was indicated that the seeds were viable.
Preferably, the tetrazole staining solution is 0.5-1.2% in mass fraction.
Preferably, the pre-wetting treatment comprises the steps of: and placing the pure seeds in an environment with 100% relative equilibrium humidity for 12-72 h, and then placing the seeds on an agar culture medium with the temperature of 17-23 ℃ and the relative equilibrium humidity of 0.8-1.2% to absorb water for 8-36 h to obtain the prewetted seeds.
Preferably, the manual cleaning of step 1) is manual kneading of the seeds.
Compared with the prior art, the invention has the beneficial effects that:
the invention provides a method for determining the viability of michelia globosa seeds, which comprises the steps of treating the michelia globosa seeds and then dyeing the michelia globosa seeds in a tetrazole dyeing solution, wherein the traditional germination experiment technology is used for determining the viability of the michelia globosa seeds for more than 1 month, and a germination experiment can underestimate the real viability of the seeds due to seed dormancy, germination conditions and the like, and the method can be completed within 40-180 hours. The method selects proper dyeing conditions such as prewetting time, dyeing solution concentration, dyeing temperature, dyeing time and the like, greatly improves the dyeing effect, and further improves the accuracy of determining the vitality of the michelia sphaera seeds. According to the method, the TTC dyeing method of the michelia sphaeroides seeds and the judgment of different conditions after dyeing are determined by the red aril manually cleaned, the pre-wetting treatment, the seed treatment after the pre-wetting, and the optimized screening of the concentration and the soaking time of the tetrazolium dyeing solution, and the determination method has great guiding significance for effectively detecting the viability of the michelia sphaeroides seeds.
Drawings
FIG. 1 is a photograph of a seed treated in example 1, showing uniform red staining of embryo and endosperm;
FIG. 2 is a photograph of the treated seed of example 2, with the embryo uniformly colored red and the endosperm non-uniformly colored red;
FIG. 3 is a photograph of a seed treated in comparative example 7, with the embryo unstained and the endosperm unevenly red;
FIG. 4 is a photograph of a seed treated in comparative example 5, showing uneven red staining of embryo and endosperm;
FIG. 5 is a photograph of the seed treated in comparative example 8, showing dark red and slightly blackened embryo and red endosperm;
FIG. 6 is a photograph of the seed treated in comparative example 9, with the embryo unstained and the endosperm stained a non-uniform red color.
Detailed Description
The invention provides a method for measuring the viability of michelia sphaerana seeds, which comprises the following steps:
1) manually cleaning red aril of the seeds to obtain pure seeds;
2) carrying out pre-wetting treatment on the pure seeds to obtain pre-wetted seeds;
3) removing endocarp from the prewetted seeds, and cutting off 2-6 mm endosperm on two sides of the seeds respectively to obtain seeds to be dyed;
4) placing the seeds to be dyed in a tetrazolium dyeing solution for dyeing treatment to obtain dyed seeds, wherein the dyeing is carried out under a dark condition, the dyeing temperature is 28-32 ℃, and the dyeing time is 20-72 hours;
5) the dyed seeds were dissected along the embryo, and when the embryo was evenly dyed red and the endosperm was dyed red, it was indicated that the seeds were viable.
In the invention, the red aril of the seed is manually cleaned. In the invention, the manual cleaning is to manually rub the seeds, and in the specific implementation process of the invention, the seeds are preferably placed on a screen mesh, rubbed by using rubber gloves, and washed under running water to remove red aril, so as to obtain pure seeds. In the present invention, the seeds are preferably mature and filled seeds. The screen cloth is preferably 21.1-21.5 cm in diameter, 5.5cm in depth and 2.5-3.5 mm in aperture, the kneading force is preferably soft, the kneading force is measured by extruding an A4 paper with two hands, the paper is extruded with two palms, and the paper cannot be easily taken away, but the paper cannot be damaged. The invention removes the red false seed coat, thus achieving the purpose of preventing the seeds from mildewing and dying. The mode of cleaning the red aril of the seeds ensures that the seeds are cleaned cleanly, and the seeds are not damaged to reduce the vitality of the seeds. The source of the seed is not particularly limited in the present invention, and commercially available seeds well known to those skilled in the art may be used.
In the present invention, when the pure seeds are subjected to the pre-wetting treatment, the specific steps of the pre-wetting treatment are preferably as follows: and placing the pure seeds in an environment with 100% relative equilibrium humidity for 12-72 h, and then placing the seeds on an agar culture medium with the temperature of 17-23 ℃ and the relative equilibrium humidity of 0.8-1.2% to absorb water for 8-36 h to obtain the prewetted seeds. The ambient temperature of 100% relative equilibrium humidity is preferably 20 ℃, the time for standing is further preferably 24 hours, and the time for water absorption is further preferably 16 hours. The method for pre-wetting the pure seeds promotes physiological and biochemical reactions of the seeds, accelerates the respiration of the seeds and enables the seeds to react with the tetrazole solution more easily.
In the invention, the endocarp of the prewetted seed is removed, and the endosperm with the diameter of 2-6 mm is cut off from the two sides of the seed. In the invention, the endocarp is removed preferably by squeezing with a pliers until the seeds crack, and then peeling off the endocarp with a knife, the method removes the endocarp from the prewetted seeds and cuts off 2-6 mm of endosperm on two sides of the seeds respectively, so that the tetrazolium staining solution is easier to permeate, and the staining time is shortened.
In the present invention, the tetrazolium staining solution is preferably 0.5% to 1.2% by weight, more preferably 1% by weight, and the tetrazolium staining solution is prepared by the following method: 0.5-1.2 g of TTC medicinal powder is weighed and dissolved in 95-120 mL of distilled water, and the dyeing temperature is preferably 28-32 ℃, and more preferably 30 ℃. The dyeing time is more preferably 48-72 h. The method of preparing the tetrazolium staining solution of the present invention is not limited, and a method known to those skilled in the art may be used. The source of the tetrazolium staining solution in the present invention is not limited, and a tetrazolium staining solution known to those skilled in the art may be used.
In the invention, when the embryo is uniformly dyed red and the endosperm is uniformly dyed red, or when the embryo is uniformly dyed red and the endosperm is dyed non-uniformly red, the two dyeing conditions can be regarded as that the seed is alive; the seed was considered non-viable when the embryo was not dyed red, the embryo was not uniformly dyed red, the embryo was uniformly dyed red and the endosperm was not dyed red, the embryo was not uniformly dyed red and the endosperm was uniformly dyed red, the embryo was not uniformly dyed red and the endosperm was dyed non-uniformly red, the embryo was not dyed red and the endosperm was uniformly dyed red, the embryo was not dyed red and the endosperm was dyed non-uniformly red, the embryo was dark red and slightly black, and the endosperm was dyed red.
The technical solution of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. It is to be understood that the described embodiments are merely exemplary of the invention, and not restrictive of the full scope of the invention. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Example 1
Collecting mature and full michelia globosa seeds with red aril, placing the michelia globosa seeds on a screen, gently kneading the seeds by using rubber gloves, and washing off the red aril under running water to obtain pure seeds; placing the pure seeds in an environment with 100% relative equilibrium humidity and 20 ℃, standing for 72h, and then placing the seeds on a 1% agar culture medium at 20 ℃ to absorb water for 24h to obtain prewetted seeds; removing black endocarps from the prewetted seeds, and cutting off 2-6 mm endosperm on two sides of the seeds respectively to obtain seeds to be dyed; soaking the seeds to be dyed in a tetrazolium dyeing solution with the mass fraction of 1%, placing the seeds in a dark incubator at 30 ℃ for 24 hours, taking out the seeds, splitting the seeds along the embryos, and observing the colors of the embryos and the endosperm;
when the embryo is uniformly dyed red and the endosperm is uniformly dyed red, or when the embryo is uniformly dyed red and the endosperm is dyed non-uniformly red, the two dyeing conditions can be regarded as that the seed is active; the seed was considered non-viable when the embryo was not dyed red, the embryo was not uniformly dyed red, the embryo was uniformly dyed red and the endosperm was not dyed red, the embryo was not uniformly dyed red and the endosperm was uniformly dyed red, the embryo was not uniformly dyed red and the endosperm was dyed non-uniformly red, the embryo was not dyed red and the endosperm was uniformly dyed red, the embryo was not dyed red and the endosperm was dyed non-uniformly red, the embryo was dark red and slightly black, and the endosperm was dyed red.
In this example, the total number of seeds in each test group was 25, and the statistically stained seeds were observed and the percentage of seeds having viability, which is viable seeds/total number of test seeds × 100%, was calculated.
Example 2
The difference between the method for determining the viability of michelia globosa seeds in this embodiment and embodiment 1 is that: the seeds to be dyed are soaked in a tetrazolium staining solution with the mass fraction of 1 percent and then placed in a dark incubator at 30 ℃ for 48 hours, and other treatment steps are the same as those in example 1.
Example 3
The difference between the method for determining the viability of michelia globosa seeds in this embodiment and embodiment 1 is that: the seeds to be dyed are soaked in a tetrazolium staining solution with the mass fraction of 1 percent and then placed in a dark incubator at 30 ℃ for 72 hours, and other treatment steps are the same as those in example 1.
Example 4
The difference between the method for determining the viability of michelia globosa seeds in this embodiment and embodiment 1 is that: the seeds to be dyed are soaked in a tetrazolium staining solution with the mass fraction of 0.5 percent and then placed in a dark incubator at 30 ℃ for 72 hours, and other treatment steps are the same as those in example 1.
Comparative example 1
The difference between the method for determining the viability of michelia globosa seeds of the comparative example and the method of example 1 is that: the seeds to be dyed are soaked in a tetrazolium staining solution with the mass fraction of 0.1 percent and then placed in a dark incubator at 30 ℃ for 24 hours, and other treatment steps are the same as those in example 1.
Comparative example 2
The difference between the method for determining the viability of michelia globosa seeds of the comparative example and the method of example 1 is that: the seeds to be dyed are soaked in a tetrazolium staining solution with the mass fraction of 0.1 percent and then placed in a dark incubator at 30 ℃ for 48 hours, and other treatment steps are the same as those in example 1.
Comparative example 3
The difference between the method for determining the viability of michelia globosa seeds of the comparative example and the method of example 1 is that: the seeds to be dyed are soaked in a tetrazolium staining solution with the mass fraction of 0.1 percent and then placed in a dark incubator at 30 ℃ for 72 hours, and other treatment steps are the same as those in example 1.
Comparative example 4
The difference between the method for determining the viability of michelia globosa seeds of the comparative example and the method of example 1 is that: the seeds to be dyed are soaked in a tetrazolium staining solution with the mass fraction of 0.1 percent and then placed in a dark incubator at 30 ℃ for 96h, and other treatment steps are the same as those in example 1.
Comparative example 5
The difference between the method for determining the viability of michelia globosa seeds of the comparative example and the method of example 1 is that: the seeds to be dyed are soaked in a tetrazolium staining solution with the mass fraction of 0.5 percent and then placed in a dark incubator at 30 ℃ for 24 hours, and other treatment steps are the same as those in example 1.
Comparative example 6
The difference between the method for determining the viability of michelia globosa seeds of the comparative example and the method of example 1 is that: the seeds to be dyed are soaked in a tetrazolium staining solution with the mass fraction of 0.5 percent and then placed in a dark incubator at 30 ℃ for 48 hours, and other treatment steps are the same as those in example 1.
Comparative example 7
The difference between the method for determining the viability of michelia globosa seeds of the comparative example and the method of example 1 is that: the seeds to be dyed are soaked in a tetrazolium staining solution with the mass fraction of 0.5 percent and then placed in a dark incubator at 30 ℃ for 96h, and other treatment methods are the same as those in example 1.
Comparative example 8
The difference between the method for determining the viability of michelia globosa seeds of the comparative example and the method of example 1 is that: the seeds to be dyed are soaked in a tetrazolium staining solution with the mass fraction of 1 percent and then placed in a dark incubator at 30 ℃ for 96 hours, and other treatment steps are the same as those in example 1.
Comparative example 9
The difference between the method for determining the viability of michelia globosa seeds of the comparative example and the method of example 1 is that: removing black endocarp from the pre-wetted seeds, and cutting off 2-6 mm endosperm on two sides of the seeds respectively; the seeds to be dyed are soaked in a tetrazolium staining solution with the mass fraction of 1 percent and then placed in a dark incubator at 30 ℃ for 24 hours, and other treatment steps are the same as those in example 1.
Comparative example 10
The difference between the method for determining the viability of michelia globosa seeds of the comparative example and the method of example 1 is that: removing black endocarp from the pre-wetted seeds, and cutting off 2-6 mm endosperm on two sides of the seeds respectively; the seeds to be dyed are soaked in a tetrazolium staining solution with the mass fraction of 1 percent and then placed in a dark incubator at 30 ℃ for 48 hours, and other treatment steps are the same as those in example 1.
Comparative example 11
The difference between the method for determining the viability of michelia globosa seeds of the comparative example and the method of example 1 is that: removing black endocarp from the pre-wetted seeds, and cutting off 2-6 mm endosperm on two sides of the seeds respectively; the seeds to be dyed are soaked in a tetrazolium staining solution with the mass fraction of 1 percent and then placed in a dark incubator at 30 ℃ for 72 hours, and other treatment steps are the same as those in example 1.
Comparative example 12
The difference between the method for determining the viability of michelia globosa seeds of the comparative example and the method of example 1 is that: removing black endocarp from the pre-wetted seeds, and cutting off 2-6 mm endosperm on two sides of the seeds respectively; the seeds to be dyed are soaked in a tetrazolium staining solution with the mass fraction of 1 percent and then placed in a dark incubator at 30 ℃ for 72 hours, and other treatment steps are the same as those in example 1.
The influence of the different methods for determining the viability of seeds of examples 1 to 4 and comparative examples 1 to 12 on the viability of seeds and the staining of seeds are shown in table 1.
The methods for determining the viability of different seeds of examples 1-4 and comparative examples 1-12 and the effects thereof are shown in Table 2.
TABLE 1 results of the viability and staining of Michelia sphenanthera seeds for different assays
As can be seen from Table 1, when the embryo is uniformly dyed red and the endosperm is uniformly dyed red, or when the embryo is uniformly dyed red and the endosperm is uniformly dyed red, both of the above dyeing cases can be regarded as the seed being viable; the seed was considered non-viable when the embryo was not dyed red, the embryo was not uniformly dyed red, the embryo was uniformly dyed red and the endosperm was not dyed red, the embryo was not uniformly dyed red and the endosperm was uniformly dyed red, the embryo was not uniformly dyed red and the endosperm was dyed non-uniformly red, the embryo was not dyed red and the endosperm was uniformly dyed red, the embryo was not dyed red and the endosperm was dyed non-uniformly red, the embryo was dark red and slightly black, and the endosperm was dyed red.
TABLE 2 results of the viability of Michelia sphenanthera seeds for different assay methods
As can be seen from table 2, if the concentration of the tetrazolium staining solution is less than 0.5%, the normal staining cannot be performed, which reduces the determination of the viability of the michelia sphaera seeds and is not favorable for the determination of the viability of the michelia sphaera seeds; when the concentration of the tetrazolium staining solution is 0.5%, the staining time is not less than 72h, and when the concentration of the tetrazolium staining solution is 1%, the staining time is 24-72 h, so that the method is favorable for measuring the vitality of the michelia figo seeds, and the vitality of the seeds can be reduced when the staining time is continuously prolonged. The endocarp is not peeled off, only 2-6 mm of the endocarp is cut off at two sides of the seeds, the tetrazole staining solution cannot normally permeate the seeds for full staining, the staining effect is poor, and the determination of reducing the seed viability is caused.
Example 5
And (3) carrying out germination tests on the seeds collected in the embodiments 2-3, counting the total number of the germinated seeds, carrying out a shearing test on the last ungerminated seeds, and judging the ungerminated reasons of the seeds, wherein the test lasts for 2 months. Each dish was seeded with 20 seeds and repeated 3 times. In this embodiment, the seed germination method is not particularly limited, and a seed germination method known in the art may be adopted, where the germination rate is the total number of germinated seeds/(total number of seeds sowed-number of damaged seeds-number of empty seeds), and the specific test results are shown in table 3:
TABLE 3 comparison of Michelia sphenanthera seed germination Rate results to seed average percent viability
As can be seen from Table 3, the seed germination test method of the present invention is similar to the seed viability percentage measured by the method of the present invention, which indicates that the method is accurate, effective, faster, simpler and more convenient.
The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.
Claims (4)
1. The method for measuring the viability of the michelia sphaerana seeds is characterized by comprising the following steps of:
1) manually cleaning red aril of the seeds to obtain pure seeds;
2) carrying out pre-wetting treatment on the pure seeds to obtain pre-wetted seeds;
3) removing endocarp from the prewetted seeds, and cutting off 2-6 mm endosperm on two sides of the seeds respectively to obtain seeds to be dyed;
4) placing the seeds to be dyed in a tetrazolium dyeing solution for dyeing treatment to obtain dyed seeds, wherein the dyeing is carried out under a dark condition, the dyeing temperature is 28-32 ℃, and the dyeing time is 20-72 hours;
5) the dyed seeds were dissected along the embryo, and when the embryo was evenly dyed red and the endosperm was dyed red, it was indicated that the seeds were viable.
2. The method of claim 1, wherein the tetrazolium staining solution is present in an amount of 0.5% to 1.2% by weight.
3. The method according to claim 1, wherein the pre-wetting treatment comprises the steps of: and placing the pure seeds in an environment with 100% relative equilibrium humidity for 12-72 h, and then placing the seeds on an agar culture medium with the temperature of 17-23 ℃ and the relative equilibrium humidity of 0.8-1.2% to absorb water for 8-36 h to obtain the prewetted seeds.
4. The method of claim 1, wherein the manual cleaning of step 1) is manual kneading of the seeds.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN116008196A (en) * | 2023-03-20 | 2023-04-25 | 中国科学院昆明植物研究所 | Method for rapidly and minimally invasively detecting activity of young garlic fruits |
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| CN116008196A (en) * | 2023-03-20 | 2023-04-25 | 中国科学院昆明植物研究所 | Method for rapidly and minimally invasively detecting activity of young garlic fruits |
| CN116008196B (en) * | 2023-03-20 | 2023-06-02 | 中国科学院昆明植物研究所 | Method for rapidly and minimally invasively detecting activity of young garlic fruits |
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