CN114747374A - Ultralow-temperature preservation method for allium chive and leek vegetable seeds - Google Patents
Ultralow-temperature preservation method for allium chive and leek vegetable seeds Download PDFInfo
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01F—PROCESSING OF HARVESTED PRODUCE; HAY OR STRAW PRESSES; DEVICES FOR STORING AGRICULTURAL OR HORTICULTURAL PRODUCE
- A01F25/00—Storing agricultural or horticultural produce; Hanging-up harvested fruit
-
- 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
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01G—HORTICULTURE; CULTIVATION OF VEGETABLES, FLOWERS, RICE, FRUIT, VINES, HOPS OR SEAWEED; FORESTRY; WATERING
- A01G22/00—Cultivation of specific crops or plants not otherwise provided for
- A01G22/35—Bulbs; Alliums, e.g. onions or leeks
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N3/00—Preservation of plants or parts thereof, e.g. inhibiting evaporation, improvement of the appearance of leaves or protection against physical influences such as UV radiation using chemical compositions; Grafting wax
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- Toxicology (AREA)
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Abstract
The invention belongs to the technical field of seed preservation, and discloses an ultralow-temperature preservation method for scallion and leek vegetable seeds, which comprises the steps of putting a proper amount of seeds into a freezing storage tube; putting the cryopreservation tube into a liquid nitrogen tank; putting the seeds subjected to ultralow temperature treatment into an artificial incubator at 40 ℃, taking out the seeds after 4 hours, and performing rewarming (thawing); carrying out germination test on the re-warmed (thawed) seeds; the germinated seeds were transferred to a plug containing the substrate for culture. The method is simple and easy, can be applied to long-term preservation of the germplasm of the allium chives and leeks vegetables, and can avoid the risk of germplasm resource loss caused by reduction of conventional preservation activity; the labor and economic costs for continuously reproducing and updating the low-activity germplasm resources in the conventional preservation are reduced; the seed source is safely stored for a long time, so that the utilization efficiency of the germplasm resources is improved; can also be applied to the preservation of allium tuberosum and leek vegetable seeds in commercial production, in particular to the safe preservation of rare seed sources.
Description
Technical Field
The invention belongs to the technical field of seed preservation, and particularly relates to an ultralow-temperature preservation method for scallion and leek vegetable seeds.
Background
The scallion and leek vegetables mainly comprise leeks, onions, leeks, scallions and the like. The vegetable is rich in sugar, vitamin c, mineral substances such as sulfur, phosphorus, iron and the like, contains bactericidal substances, has the functions of promoting appetite, seasoning, removing fishy smell, treating medical treatment and the like, and is an important special vegetable for both medicine and vegetables.
The allium tuberosum and leek vegetables are various in types, the genetic diversity of germplasm resources is very rich, and the resources are mainly stored in a seed form and serve for breeding and industrial development. The allium chive vegetable seeds belong to short-lived seeds, and the vitality of the allium chive vegetable seeds is obviously reduced in 1-2 years at normal temperature. Therefore, preservation of these resources is mainly performed in low-temperature germplasm banks. However, researches find that the preservation of the germplasm resources in low-temperature preservation is not ideal, the germplasm resources are obviously reduced after 3 to 5 years, and the germination rate of seeds is seriously influenced. In practical implementation, the germplasm resource repository has to consume a large amount of manpower and material resources to continuously monitor the vitality of the resources, and the resources are updated and rejuvenated every 2 to 5 years. How to safely preserve these national strategic resources for a long time faces a great challenge. Therefore, it is of great significance to research a method for safely preserving the germplasm resources for a long time.
The ultra-low temperature preservation is an important component of the in vitro preservation technology, has the advantages of environmental protection, cost saving, convenience and the like, and is an ideal method for preserving the plant germplasm resources for a long time. The ultra-low temperature preservation technology is a method for effectively preserving germplasm resources for a long time at present, and refers to a biological technology for preserving planting resources at the ultra-low temperature of below 80 ℃ below zero, wherein liquid nitrogen is the most common, and under the ultra-low temperature environment of 196 ℃ below zero (liquid nitrogen), the substance metabolism and the growth activity of almost all living cells of plant seeds are in a relatively stable biological state. This method allows the genetic stability of the biological material to be maintained without losing its morphogenic potential. The ultra-low temperature preservation technology can preserve and rescue species, can avoid the occurrence of species mutation, and is an important method for stably preserving plant species resources and precious experimental materials for a long time. The liquid nitrogen ultra-low temperature preservation technology is developed from the beginning to successfully store plant materials to the present, and is recognized as one of the best methods for the long-term preservation of germplasm. The plant seeds can stop cell metabolism in the environment of liquid nitrogen ultralow temperature, so that the cell activity and morphology can be stably preserved theoretically. In the early stage of cryopreservation, freezing preservation techniques are mostly used, and mainly include a direct freezing method, a stepwise freezing method, a vitrification method, a dry freezing method, an embedding dehydration method, and the like. Until now, some degree of germination was obtained after freezing snapdragon pollen to-180 ℃ in 1922 h.e.knowlton. This was the earliest report of cryopreservation of plant germplasm. In 1956, Japanese scientist A.Sakai reported that cold-resistant mulberry branches survive cryopreservation after freeze dehydration for the first time. In 1968 r.s.quatrano reported that flax cultured cells were resistant to low temperatures of-50 ℃ after pretreatment with (DMSO). In 1971, R.Latta reported that carrot suspension cells were successfully preserved in liquid nitrogen (-196 ℃), and that embryoid bodies were regenerated. In 1976, M.Seiburt reports the success of ultralow-temperature preservation of the stem tips of the moschus brachyotus for the first time, and indicates that the effect of 2-3 pairs of leaf primordia is the best, and then the ultralow-temperature preservation technology is gradually and deeply researched in the aspect of plant germplasm resource preservation, and the application is continuously expanded. Nearly thousands of plants are successfully subjected to ultralow temperature preservation, and some plant germplasm resources are subjected to large-scale ultralow temperature preservation; such as apple, mulberry dormant buds and potato stem tips. Zhengguang planting et al succeeded in ultralow temperature preservation of medicinal plant callus and its suspension cultured cells in 1983, and became the pioneer of the ultralow temperature preservation research of plant germplasm resources in China. In 1989, the vitrification method was used for the first time to successfully cryopreserve asparagus somatic embryos and mint suspension cultures by Uragami et al and Langis et al. In 1992, Niino et al, using an embedding drying method to preserve the isolated stem tips of 5 apple varieties, obtained higher survival rates. The research in China is started late, and the in vitro stem tip of the apple is preserved by embedding, drying and ultralow temperature technology in 1998 by Zhao Yanhua et al, which is successful. Research also finds that in 2015, people of Zenglin et al have performed liquid nitrogen ultralow temperature preservation experimental exploration on the alpinia seeds, and the liquid nitrogen ultralow temperature preservation of the alpinia seeds is proved to be feasible. 2016 Songhong et al, has reduced soluble protein content in seed of Cordyceps cicadae Miquel after ultralow temperature freezing, Peroxidase (POD) activity, and superoxide dismutase (SOD) activity of stem tip of flos Chrysanthemi. Although there are a number of methods for cryopreservation of plant material, there is no method for the storage of all plant material to date. Therefore, in order to properly preserve plant materials, it is necessary to select an appropriate method according to the characteristics of the materials.
In summary, the ultra-low temperature preservation technology can realize the long-term safe preservation of seeds, but key technical research needs to be developed aiming at the characteristics of different crops, so that a corresponding ultra-low temperature preservation method is established. At present, no report is found on an ultralow-temperature preservation method applied to allium chive and leek vegetable seeds.
Through the above analysis, the problems and defects of the prior art are as follows: scallion and leek vegetables belong to short-lived germplasm and face a great challenge to long-term safe storage; how to simply and efficiently preserve the germplasm resources of the vegetable vegetables lacks technical reference; an ultralow-temperature preservation technology aiming at allium chive and leek vegetable germplasm resources is not available; whether the physiological indexes of the scallion and leek seeds are changed after being stored at ultralow temperature and the change rule are unclear.
Disclosure of Invention
Aiming at the problems in the prior art, the invention provides an ultralow-temperature preservation method for allium chive seeds.
The invention is realized in such a way that the ultralow-temperature preservation method of the scallion and leek seeds comprises the following steps:
step one, taking a proper amount of seeds for treatment, and putting the seeds into a freezing storage tube;
taking appropriate amount of seeds, placing the seeds in a culture dish, and soaking the seeds in cryoprotectant PVS2 (soaking for 0min,20min, 30min, 40min) with a dropper. (PVS 2): 30% glycerol + 15% ethylene glycol + 15% dimethylsulfoxide +0.4 mol. L sucrose. Taking out the seeds, wiping the residual PVS2 on the surfaces of the seeds by using filter paper, and putting the seeds into a freezing storage tube;
Putting the freezing tube into a liquid nitrogen tank;
and step three, performing rewarming (thawing) on the seeds after the ultralow temperature treatment, and standing for 4 hours, 10 hours, 16 hours and 24 hours at four different levels of temperature of 4 ℃, 25 ℃, 30 ℃ and 40 ℃ according to a test method designed in an orthogonal table (table 1).
Step four, carrying out a germination test on the rewarming (thawing) seeds;
and step five, transferring the germinated seeds to a plug tray containing a substrate for culture.
Further, the germination test step in the fourth step comprises:
preparing a transparent plastic culture dish, soaking the sterilized filter paper in distilled water, and putting the filter paper into the culture dish;
and putting the seeds which are subjected to rewarming (thawing) at different time and temperature into an artificial incubator at 22 ℃ for seed germination experiments, wherein 50 seeds are obtained in each group, and the steps are repeated for three times.
By combining the technical scheme and the technical problem to be solved, the invention provides the simple method for storing the scallion and leek vegetable seeds for a long time; the large-scale safe preservation of the allium tuberosum and leek vegetable germplasm resources can be realized; greatly reduces the material resource consumption of regularly breeding and improving the seed source activity.
Drawings
FIG. 1 is a flow chart of the ultralow temperature preservation method of leek seeds provided by the embodiment of the invention;
FIG. 2 is a graph illustrating the effect of various factor curves provided by an embodiment of the present invention;
fig. 3 is a first diagram illustrating the effect of the experiment process according to the embodiment of the present invention.
Fig. 4 is a diagram of an effect of the experiment process provided by the embodiment of the invention.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is further described in detail with reference to the following embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and do not limit the invention.
As shown in figure 1, the ultralow-temperature preservation method for allium chive seeds provided by the embodiment of the invention comprises the following steps:
s101, taking a proper amount of seeds and putting the seeds into a freezing storage tube;
s102, putting the cryopreservation tube into a liquid nitrogen tank;
s103, performing rewarming (thawing) on the seeds after the ultralow temperature treatment;
s104, performing a germination test on the rewarmed (thawed) seeds;
s105, transferring the germinated seeds to a plug tray containing a substrate for culture.
The step S101 of taking a proper amount of seeds for processing and putting the seeds into a freezing storage tube comprises the following steps:
taking appropriate amount of seeds, placing the seeds in a culture dish, and soaking the seeds in cryoprotectant PVS2 (soaking for 0min, 20min, 30min, 40min) with a dropper. (PVS 2): 30% glycerol + 15% ethylene glycol + 15% dimethylsulfoxide +0.4 mol. L sucrose. Taking out the seeds, wiping the residual PVS2 on the surfaces of the seeds by using filter paper, and putting the seeds into a freezing storage tube;
And S103, rewarming (thawing) the seeds after the ultralow temperature treatment, and standing for 4h, 10h, 16h and 24h at four different levels of temperature of 4 ℃, 25 ℃, 30 ℃ and 40 ℃ according to a test method designed in an orthogonal table (table 1).
Further, the germination testing step in step S104 includes:
preparing a transparent plastic culture dish, soaking the sterilized filter paper in distilled water, and putting the filter paper into the culture dish;
and putting the seeds which are subjected to rewarming (thawing) at different time and temperature into an artificial incubator at 22 ℃ for seed germination experiments, wherein 50 seeds are obtained in each group, and the steps are repeated for three times.
The embodiment of the invention achieves some obvious effects in the process of research and development or use, and has great advantages compared with the prior art.
Advantage 1:
the seeds preserved by the method of the invention well keep the original vitality and can successfully grow and develop into complete plants, thereby achieving the effect of preserving the seeds for a long time;
advantage 2: large-scale experimental research proves that the method for preserving the leek and the scallion seeds at the ultralow temperature by using liquid nitrogen is very reliable, and the same effect is obtained on the ultralow temperature preservation of the leek and the onion leek seeds;
advantage 3: by comparing the optimal ultralow-temperature preservation conditions of different germplasms, the liquid nitrogen ultralow-temperature preservation method disclosed by the invention has good applicability to different provenances; advantage 4 relates to a technical method for researching and researching ultralow temperature preservation of scallion and leek vegetable seeds successfully by the method.
The following is described in connection with data, graphs, etc. of the experimental procedure.
1. The material and the method are as follows:
1.1 materials:
the tested materials are 4 chive and leek vegetable germplasm resources, namely chive, scallion and onion.
1.2 Experimental methods:
1.2.1 selecting experimental materials:
randomly selecting 10 parts of leek germplasm, 10 parts of scallion germplasm, 5 parts of leek germplasm and 5 parts of onion and leek germplasm.
1.2.2 design of orthogonal experiments:
orthogonal experimental design refers to a multi-factor and multi-level experimental design method. When the number of factors involved in the test is 3 or more than 3, the test workload becomes large and the implementation is difficult. The orthogonal test is a good choice for the difficulty, and can achieve the equivalent result with a large number of comprehensive tests with the least test times, so the invention applies the orthogonal table design test. The success of cryopreservation is determined by the material characteristics, the preculture method, the cryoprotectant, the freezing method and other factors. Therefore, factors influencing the ultra-low temperature preservation of the seeds are PVS2 processing time (factor A), thawing temperature (factor B) and thawing time (factor C). Each factor set 4 treatment levels. Factor A is PVS2 soaking (soaking)Bubble A 1(0min)、A2(20min)、A3(30min)、A4(40min)；B1(4℃)、 B2(25℃)、B3(30℃)、B4(40℃)；C1(4h)、C2(10h)、C3(16h)、 C4(24h) .1. the Common quadrature design L16(45) Orthogonal table (table 1), a total of 16 tests were performed, and the ratio of the germination rates of seeds treated in different combinations (normal seed germination rate/germination rate of seeds after cryopreservation) was used as the orthogonal test result. Statistical analysis was performed by SPSS software.
TABLE 1 cryopreservation method orthogonal test
1.2.3 seed cryopreservation method:
the method comprises the following general steps of selection and preparation of materials, pretreatment, cooling and freezing, thawing, survival identification of the materials and genetic stability analysis. The experiment adopts direct freezing method and vitrification method to preserve scallion and leek seeds at ultra-low temperature.
Direct freezing method:
step one, taking a proper amount of seeds and putting the seeds into a freezing storage tube;
putting the freezing tube into a liquid nitrogen tank;
step three, performing rewarming (thawing) on the seeds after ultralow temperature treatment;
a vitrification method:
step one, taking a proper amount of seeds and placing the seeds in a culture dish;
step two, soaking the seeds by using a dropper to suck the cryoprotectant PVS 2. (PVS 2): 30% of glycerol, 15% of ethylene glycol, 15% of dimethyl sulfoxide and 0.4mol of L-sucrose;
step three, taking out the seeds and sucking residual PVS2 on the surfaces of the seeds by using filter paper
Step four, taking a proper amount of seeds and putting the seeds into a freezing storage tube
Step five, putting the freezing tube into a liquid nitrogen tank
Step six, performing rewarming (thawing) on the seeds after ultralow temperature treatment;
1.2.4 seed Germination test
A seed germination test is carried out by adopting a culture dish filter paper method, a transparent plastic culture dish is prepared, and the sterilized filter paper is soaked in distilled water and placed in the culture dish. And putting the seeds which are subjected to rewarming (thawing) for different time and temperature into a 22 ℃ artificial incubator for carrying out a traditional seed germination experiment, wherein 50 seeds are obtained in each group, and the steps are repeated for three times. The germination rate was counted on day 12 after cultivation according to the national standard "crop seed test protocol".
2. Results and analysis:
2.1 analysis of orthogonal test results
The results of the analysis of variance (table 2) by designing an orthogonal test showed that the F values for factor a and factor C were less than the F threshold, with significant differences at a p <0.05 level. From this, it was found that the factor a (PVS2 treatment time) and the factor C (rewarming (thawing) time) significantly affect the test results, and the factor B (rewarming (thawing) temperature) does not significantly affect the test results. The main factors that influence the test results can be determined as factor a (PVS2 treatment time) and factor C (rewarming (thawing) time).
TABLE 2 orthogonal test design ANOVA TABLE
And (3) drawing a curve effect diagram of each factor by taking the level of each factor as an abscissa and taking the average value of the test index as an ordinate (figure 2). The trend chart can more intuitively see the trend of the test index changing along with the factor level. The results showed that the germination percentage ratio gradually decreased with the increase of the PVS2 treatment time, the germination percentage ratio gradually increased with the increase of the temperature at the time of rewarming (thawing), and the germination percentage ratio gradually decreased with the increase of the rewarming (thawing) time. In this test, the larger the test index, the better. It can be determined that the main factor affecting the experimental results is the PVS2 treatment time, and the optimal level is 0h treated with PVS 2. However, the time for the factor rewarming (thawing) and the temperature curve required by the factor rewarming (thawing) are relatively flat, and the two factor levels have little influence on the test result.
30 parts of different test materials were randomly selected from the test materials. The tests were performed according to 16 different combinations of tests in the orthogonal table (table 1), i.e. PVS2 solution was treated for different times, put into liquid nitrogen for 24h, taken out and re-warmed (thawed) at different temperatures for different gradient durations. And immediately carrying out a germination test on the seeds after the re-warming (thawing) is finished, counting the germination rate, and comparing the germination rate with the original germination rate of the seeds which are not frozen by liquid nitrogen, wherein the ratio of the two is the result of the orthogonal test. The larger the ratio, the better the preservation effect of the test carried out with this test combination. The average value k of the levels of different factors is compared by the visual analysis method in Table 1, and the optimal level combination of the test is A 1B4C1I.e. the composite material is not processed by PVS2 (cryoprotectant) and is reheated (thawed) at the constant temperature of 40 ℃ for 4 h.
Wherein, fig. 3 and fig. 4 are effect diagrams of the experiment process provided by the embodiment of the invention.
The above description is only for the purpose of illustrating the present invention and the appended claims are not to be construed as limiting the scope of the invention, which is intended to cover all modifications, equivalents and improvements that are within the spirit and scope of the invention as defined by the appended claims.
Claims (10)
1. The ultralow-temperature preservation method for the allium fistulosum and leek seeds is characterized by comprising the following steps of:
firstly, taking seeds for processing, and putting the seeds into a freezing storage tube;
putting the cryopreservation tube into a liquid nitrogen tank;
thirdly, re-warming the seeds after ultralow temperature treatment;
step four, carrying out a germination test on the rewarmed seeds;
and step five, transferring the germinated seeds to a plug tray containing a substrate for culture.
2. The cryopreservation method of Allium tuberosum seeds of claim 1, wherein in step one, the seeds are taken by direct freezing and placed in a freezing tube.
3. The cryopreservation method of allium chive seeds as claimed in claim 1, wherein in step one, the seeds are taken by direct freezing and put into a freezing tube.
4. The method for cryopreservation of allium chive seeds as claimed in claim 1 wherein in step one the seeds are treated by vitrification into a frozen tube.
5. The cryopreservation method of Allium tuberosum seeds of claim 4 wherein the vitrification comprises: taking a proper amount of seeds, placing the seeds in a culture dish, and then sucking a cryoprotectant PVS2 by using a dropper to soak the seeds; the seeds were removed and the residual PVS2 on the surface of the seeds was wiped dry with filter paper and placed in a cryopreservation tube.
6. The cryopreservation method of Allium tuberosum seeds of claim 5, wherein the protectant PVS2 is soaked for 20min, 30min, 40 min.
7. The cryopreservation method of allium tuberosum seeds of claim 5, wherein the protectant PVS2 is: (PVS 2): 30% glycerol + 15% ethylene glycol + 15% dimethylsulfoxide +0.4 mol. L sucrose.
8. The method for ultralow temperature preservation of leek seeds as claimed in claim 1, wherein in the step three, the seeds after ultralow temperature treatment are subjected to temperature recovery, and the seeds are placed at four constant temperatures of different gradients of 4 ℃, 25 ℃, 30 ℃ and 40 ℃ for 4h, 10h, 16h and 24 h.
9. The cryopreservation method of allium chive seeds as claimed in claim 1, wherein the germination test step in step four comprises:
preparing a transparent plastic culture dish, soaking sterilized filter paper in distilled water, and putting the filter paper into the culture dish;
and putting the seeds which are rewarmed for different time and temperature for carrying out a seed germination experiment.
10. The cryopreservation method of Allium tuberosum seeds of claim 9, wherein the germination test is performed in an artificial incubator at 22 ℃ with 50 seeds per group, and is repeated three times.
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| KR20110051544A (en) * | 2009-11-10 | 2011-05-18 | 대한민국(관리부서 : 산림청 국립산림과학원장) | Preservation method of japanese cedar seed in cryopreservation |
| CN104222072A (en) * | 2014-08-27 | 2014-12-24 | 中国农业科学院麻类研究所 | Vitrification ultralow-temperature preservation method of flax seeds |
| CN111170780A (en) * | 2020-01-07 | 2020-05-19 | 合肥绿农肥业有限责任公司 | Nutritional seed soaking liquid for improving germination rate and seedling activity of Chinese chives and application method thereof |
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| KR20110051544A (en) * | 2009-11-10 | 2011-05-18 | 대한민국(관리부서 : 산림청 국립산림과학원장) | Preservation method of japanese cedar seed in cryopreservation |
| CN104222072A (en) * | 2014-08-27 | 2014-12-24 | 中国农业科学院麻类研究所 | Vitrification ultralow-temperature preservation method of flax seeds |
| CN111170780A (en) * | 2020-01-07 | 2020-05-19 | 合肥绿农肥业有限责任公司 | Nutritional seed soaking liquid for improving germination rate and seedling activity of Chinese chives and application method thereof |
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