A seed treatment method for improving the drought and cold tolerance of pumpkins through nanomaterials

Abstract

The present invention belongs to the technical field of crop planting, and particularly relates to a seed treatment method for promoting the germination of pumpkin seeds and the growth of seedlings under drought stress or low-temperature stress by using nanomaterials. The method of the present invention mainly includes the following steps: 1) preparing a silver nanosol suspension; 2) cleaning and disinfecting pumpkin seeds; 3) soaking pumpkin seeds with the silver nanosol suspension; 4) seed germination. Before the germination of pumpkin seeds, by using a silver nanosol suspension with a certain concentration for seed treatment, the germination speed, seed vigor and seedling growth of pumpkin seeds under drought stress or low-temperature stress can be significantly improved. The operation process of the present invention is simple, the promotion effect is remarkable, the cost is low, and it is easy to popularize, which has important significance for the innovation of pumpkin germplasm resources and the breeding of durable resistance.

Description

Technical Field

[0001] The present invention belongs to the technical field of crop cultivation, and particularly relates to a seed treatment method using nanomaterials to promote the germination of pumpkin seeds and the growth of seedlings under drought stress or low temperature stress. Background Art

[0002] Pumpkin ( Cucurbita moschata ) has a cultivation history of more than 500 years in China and is a traditional crop that can be used as both vegetables and grains. Currently, China is the largest producer and consumer of pumpkins, and they are planted all over the country with a high yield. Pumpkins are highly nutritious, rich in starch, fat, reducing sugar, amino acids, vitamins, minerals and other substances. In addition to being an important vegetable in summer and autumn, they also have uses such as ornamental, feed, and seed use, with broad application prospects.

[0003] With the increasing intensification of climate change, crops are facing more and more environmental stresses, such as drought, high temperature, low temperature, etc. Different types of environmental stresses will have varying degrees of impact on the yield and quality of crops. The pumpkin planting areas in China are widely distributed, but mainly in the northern regions. There are often low-temperature cold damages in early spring and winter freeze damages occur from time to time, and the heat conditions fluctuate greatly with the seasons. Pumpkins prefer warm temperatures, and the suitable growth temperature is 25 - 30 °C. Encountering cold climates will seriously affect their germination and growth. Research shows that when the temperature is 15 °C, the initial germination time of pumpkin seeds is 4 days; when the temperature is 25 °C, the initial germination time is 2 days. As the temperature continues to rise, indicators such as the germination rate and germination index of the seeds first increase and then decrease, with the lowest values at 15 °C. It can be seen that low-temperature stress inhibits the germination of pumpkin seeds. In addition, drought stress is also one of the main limiting factors in pumpkin production. Drought stress will inhibit the photosynthesis of plants, disrupt the respiration of plants, cause abnormal metabolism of plants, and even lead to the cessation of plant growth, seriously affecting the growth and development of plants. Research shows that drought stress has obvious effects on indicators such as the germination rate, germination potential, germination index, vigor index, antioxidant enzyme activity of pumpkin seeds, as well as pumpkin yield and quality. Therefore, in the context of future climate change, in order to meet the growing demand for pumpkin supply, it is urgent to improve the drought tolerance and cold tolerance of pumpkins.

[0004] In recent years, nanotechnology has shown great potential in the field of agriculture. For example, carbon nanomaterials can promote the absorption of water by seeds, accelerate seed germination, and increase the germination rate of seeds. "Nano seed treatment technology" as a new way to improve seed quality can effectively promote seed germination, enhance seedling vitality, and improve plant stress resistance. Nanosilver (AgNPs) has become one of the most widely used and fastest-growing nanomaterials with its excellent chemical stability, antibacterial properties, catalytic properties, etc., and is widely used in various fields. Studies have shown that Ag NPs can catalyze the production of reactive oxygen species (ROS) in cells, and ROS, as a signaling molecule, can combine with many other signaling pathways to trigger the system defense network and increase the resistance of crops to environmental stress. For example, under salt stress, nanosilver treatment can increase the germination rate, root length, seedling fresh weight and dry weight of tomatoes, and the related gene expression patterns also indicate that nanosilver may be involved in the regulatory response of stress.

[0005] Therefore, the present invention proposes a method for treating pumpkin seeds under drought stress or low temperature stress with nanosilver materials to promote their germination and seedling growth, achieving excellent technical effects, solving the problems in the pumpkin agricultural production process, and providing scientific basis and technical support for the application of nanomaterials in agriculture under the background of climate change. Summary of the invention

[0006] Based on the above problems, the purpose of the present invention is to provide a seed treatment method using nanomaterials to promote pumpkin seed germination and seedling growth under drought stress or low temperature stress. The present invention has a simple operation process, significant promotion effect, low cost, and is easy to promote, which is of great significance to pumpkin germplasm resource innovation and durable resistance breeding.

[0007] To achieve the above object, the present invention provides the following technical solutions.

[0008] One aspect of the present invention provides a seed treatment method for improving drought resistance and cold resistance of pumpkin by using nano materials, the method comprising the following steps:

[0009] 1) Prepare nanosilver suspension: Add nanosilver particles into deionized water and ultrasonically disperse them in an ultrasonic cleaner;

[0010] 2) Clean and disinfect pumpkin seeds: After removing impurities and shrunken seeds from pumpkin seeds, wash them with deionized water, disinfect them with sodium hypochlorite solution, and finally wash them with deionized water until they are odorless;

[0011] 3) Soaking pumpkin seeds with nanosilver suspension: Add nanosilver suspension to pumpkin seeds and place them in a constant temperature oscillating incubator for soaking and cultivation. After the soaking and cultivation, pour out the solution, wash the nanosilver suspension remaining on the surface of the seeds with deionized water, and dry the water to obtain the treated seeds;

[0012] 4) Seed germination: The treated seeds were germinated using the on - paper germination method and then cultured in an artificial climate incubator. The number of germinated seeds was recorded with the criterion that the radicle exceeded half of the seed length.

[0013] Preferably, in step 1, the chemical formula of the silver nanoparticles is Ag NPs, and the particle size is 25 nm.

[0014] Preferably, the ultrasonic treatment conditions in step 1 are: power 100 w, frequency 40 kHz, and time 30 min.

[0015] Preferably, the concentration of the silver nano - suspension in step 1 is 40 mg / L.

[0016] Preferably, in step 2, the disinfection is carried out using a 5% sodium hypochlorite solution for 10 min.

[0017] Preferably, the conditions of the constant - temperature shaking incubator in step 3 are set as: light - proof, temperature 25 °C, rotation speed 100 rpm, and soaking culture time 10 h.

[0018] Preferably, the specific steps of the on - paper germination method in step 4 are: placing filter paper in a petri dish, adding 5 ml of deionized water, and putting 15 pumpkin seeds in each petri dish.

[0019] Preferably, the culture conditions of the artificial climate incubator used in the seed treatment method for improving the drought tolerance of pumpkins in step 4 are: temperature 25 °C, humidity 70%, time 5 d, light - proof culture; the culture conditions of the artificial climate incubator used in the seed treatment method for improving the cold tolerance of pumpkins are: temperature 15 °C, humidity 70%, time 15 d, light - proof culture.

[0020] Compared with the prior art, the beneficial effects achieved by the present invention are: Before the germination of pumpkin seeds, by treating the seeds with a silver nano - suspension of a certain concentration, the germination speed, seed vigor, and seedling growth of pumpkin seeds under drought stress or low - temperature stress can be significantly improved. The operation process of the present invention is simple, the promotion effect is remarkable, the cost is low, and it is easy to popularize, which has important significance for the innovation of pumpkin germplasm resources and the breeding of durable resistance. Description of the Drawings

[0021] Figure 1 Shows the effect of Ag NPs seed treatment on the germination speed of pumpkin seeds under drought stress;

[0022] Figure 2 Shows the effect of Ag NPs seed treatment on the vigor index of pumpkin seeds under drought stress;

[0023] Figure 3Effect of Ag NPs seed treatment on the germination bud length of pumpkin seeds under drought stress;

[0024] Figure 4 Effect of Ag NPs seed treatment on the germination root length of pumpkin seeds under drought stress;

[0025] Figure 5 Effect of Ag NPs seed treatment on the germination speed of pumpkin seeds under low temperature stress;

[0026] Figure 6 Effect of Ag NPs seed treatment on the vigor index of pumpkin seeds under low temperature stress;

[0027] Figure 7 Effect of Ag NPs seed treatment on the germination bud length of pumpkin seeds under low temperature stress;

[0028] Figure 8 Effect of Ag NPs seed treatment on the germination root length of pumpkin seeds under low temperature stress. Embodiment

[0029] The following will combine the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all of the embodiments. Based on the embodiments in the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts shall fall within the protection scope of the present invention. Example

[0030] 1. Material and solvent preparation

[0031] Test seeds: Pumpkin seeds (Longyuan Lixiang), 100 g.

[0032] Nanomaterials: Silver nanoparticles (AgNPs), particle size 25 nm.

[0033] Mass concentration setting of drought stress PEG solution: H-unstress: 0%; Ag NPs-unstress: 0%; H-10%PEG: 10%; Ag NPs-10%PEG: 10%.

[0034] Low temperature stress temperature setting: H-15°C: 15°C; Ag NPs-15°C: 15°C.

[0035] 2. Seed treatment process:

[0036] 1) Preparation of silver nanoparticles suspension: Silver nanoparticles (AgNPs, 25 nm) were dispersed in deionized water by ultrasonic vibration (100 W, 40 kHz) for 30 min to prepare a 40 mg / L silver nanoparticles suspension.

[0037] 2) Cleaning and disinfecting pumpkin seeds: After removing impurities and shriveled seeds from the pumpkin seeds, 100 g of high-quality pumpkin seeds with uniform size, plump grains, and consistent color were weighed. The pumpkin seeds were first washed with deionized water, then disinfected with a 5% sodium hypochlorite solution for 10 min, and finally rinsed with deionized water 6 - 7 times until odorless, and then dried with absorbent paper.

[0038] 3) Seed treatment: The cleaned and disinfected pumpkin seeds were evenly divided into two parts and placed in 500 ml glass screw-cap reagent bottles with blue caps respectively. 500 ml of Ag NPs solutions with different concentrations (0 mg / L and 40 mg / L respectively) were added, and then placed in a constant temperature dark shaking incubator at 25℃ for immersion culture, with the conditions set at 25℃ and 100 rpm. After 10 h of immersion, the solution was poured out, and the seeds were washed 5 times with deionized water and dried with absorbent paper.

[0039] 4) Seed germination: The treated seeds were germinated by the paper germination method. The specific process of the drought stress experiment was as follows: Filter paper was placed in a petri dish, and 5 ml of PEG solutions with different mass concentrations (0% and 10% respectively) were added. 15 pumpkin seeds were placed in each petri dish. Then it was placed in an artificial climate incubator (25℃, humidity 70%) for dark culture for 5 d, and the number of germinated seeds was recorded with the radicle exceeding half of the seed length as the germination standard. After 5 days, the germination ended, and the shoot length and root length were measured. The experiment had 5 replicates. The specific process of the low-temperature stress experiment was as follows: Filter paper was placed in a petri dish, and 5 ml of deionized water was added. 15 pumpkin seeds were placed in each petri dish. Then it was placed in an artificial climate incubator (15℃, humidity 70%) for dark culture for 15 d, and the number of germinated seeds was recorded with the radicle exceeding half of the seed length as the germination standard. After 15 days, the germination ended, and the shoot length and root length were measured. The experiment had 5 replicates.

[0040] 3. Measurement method

[0041] Shoot length: Measure the shoot length of normally germinated seeds on the fifth day under drought stress.

[0042] Measure the shoot length of normally germinated seeds on the fifteenth day under low-temperature stress.

[0043] Root length: Measure the root length of normally germinated seeds on the fifth day under drought stress.

[0044] Measure the root length of normally germinated seeds on the fifteenth day under low-temperature stress.

[0045] Germination rate: Under drought stress, it is (the number of seeds germinated normally on the fifth day / total number) × 100%

[0046] Under low temperature stress, it is (the number of seeds germinated normally on the fifteenth day / total number) × 100%

[0047] Vigor index: (bud length + root length) × germination rate

[0048] 4. Experimental results

[0049] The experimental results are as shown in the appendix Figures 1-8 below. Among them, in the figures, H-unstress refers to being treated with a silver nanoparticle suspension at a concentration of 0 mg / L for 10 h under the condition that the PEG mass concentration is 0%; Ag NPs -unstress refers to being treated with a silver nanoparticle suspension at a concentration of 40 mg / L for 10 h under the condition that the PEG mass concentration is 0%; H-10% PEG refers to being treated with a silver nanoparticle suspension at a concentration of 0 mg / L for 10 h under the condition that the PEG mass concentration is 10%; Ag NPs -10% PEG refers to being treated with a silver nanoparticle suspension at a concentration of 40 mg / L for 10 h under the condition that the PEG mass concentration is 10%; H-15°C refers to being treated with a silver nanoparticle suspension at a concentration of 0 mg / L for 10 h under the condition that the temperature is 15°C; Ag NPs -15°C refers to being treated with a silver nanoparticle suspension at a concentration of 40 mg / L for 10 h under the condition that the temperature is 15°C.

[0050] Referring to the appendix Figures 1-4 It can be seen that under 10% PEG drought stress: Compared with the control treatment group H-unstress, the vigor index of pumpkin seeds decreased from 920.0 cm% in the control to 386.0 cm%, the bud length decreased from 1.44 cm in the control to 0.36 cm, and the root length decreased from 7.70 cm in the control to 4.06 cm. The germination speed of pumpkin seeds decreased significantly, indicating that drought stress has a serious impact on seed germination and seedling growth.

[0051] Under normal conditions: Ag NPs-unstress. Compared with the control treatment group H-unstress, the vigor index of pumpkin seeds increased from 920.0 cm% in the control to 994.0 cm%, the bud length increased from 1.44 cm in the control to 1.82 cm, and the root length increased from 7.70 cm in the control to 8.41 cm, significantly promoting pumpkin seed germination and seedling growth. Under 10% PEG drought stress: AgNPs-10% PEG. Compared with the control treatment group H-10% PEG, the vigor index of pumpkin seeds increased from 386.0 cm% in the control to 532.0 cm%, the bud length increased from 0.36 cm in the control to 0.42 cm, and the root length increased from 4.06 cm in the control to 5.41 cm, significantly increasing the germination speed of pumpkin seeds under drought stress and alleviating the inhibitory effect of drought stress on seedling growth.

[0052] Refer to the appendix Figures 5-8 It can be seen that, similar to drought stress, low temperature stress can also have an adverse effect on seed germination and seedling growth. Under low temperature stress at 15 °C: Ag NPs-15 °C. Compared with the control treatment group H-15 °C, the vigor index of pumpkin seeds increased from 957.0 cm% in the control to 1097.0 cm%, the bud length increased from 0.96 cm in the control to 1.12 cm, and the root length increased from 9.05 cm in the control to 10.30 cm, significantly increasing the germination speed of pumpkin seeds under low temperature stress and alleviating the damage of low temperature stress to pumpkin seedlings.

[0053] In summary, the present invention provides a seed treatment method for improving the drought tolerance and cold tolerance of pumpkins. By treating pumpkin seeds with a 40 mg / L silver nanoparticle solution for 10 h under constant temperature and dark conditions, the vigor index, bud length, and root length of pumpkin seeds under drought stress (drought degree: 10% PEG) and low temperature stress (low temperature 15 °C) are significantly increased, and the drought tolerance and cold tolerance of pumpkin seeds are significantly improved. Through silver nanoparticle seed treatment, the germination of pumpkin seeds and seedling growth under drought stress or low temperature stress can be effectively promoted. The operation of the present invention is simple, the effect is remarkable, the cost is low, and it is easy to promote, filling the technical gap of the application of silver nanoparticle technology in pumpkin seeds, and is of great significance for solving the problem of environmental stress encountered in the pumpkin agricultural production process and the innovation of pumpkin germplasm resources, providing a scientific basis and technical support for the application of nanomaterials in agriculture under the background of climate change.

[0054] Details not described in the present invention are all well-known technologies to those skilled in the art.

[0055] Finally, it should be noted that the above specific embodiments are only used to illustrate the technical solutions of the present invention and not to limit them. Although the present invention has been described in detail with reference to the embodiments, those of ordinary skill in the art should understand that the technical solutions of the present invention can be modified and equivalently replaced without departing from the spirit and scope of the technical solutions of the present invention, and they should all be covered within the scope of the claims of the present invention.

Claims

1. A seed treatment method for improving the drought and cold tolerance of pumpkins by nanomaterials, the method comprising the following steps: 1) Prepare a silver nanometer suspension: Add silver nanometer particles into deionized water and place it in an ultrasonic cleaner for ultrasonic dispersion; 2) Clean and disinfect pumpkin seeds: After removing impurities and shriveled seeds from pumpkin seeds, first wash them with deionized water, then disinfect them with sodium hypochlorite solution, and finally wash them with deionized water until odorless; 3) Soak pumpkin seeds with the silver nanometer suspension: Add the silver nanometer suspension to pumpkin seeds, place them in a constant temperature shaking incubator for soaking culture. After completion, pour out the solution, wash the residual silver nanometer suspension on the seed surface with deionized water, and dry the water to obtain the treated seeds; 4) Seed germination: Use the paper germination method to germinate and culture the treated seeds, and then place them in an artificial climate incubator for culture. Record the number of germinated seeds with the radicle exceeding half of the seed length as the germination standard; In step 1, the chemical formula of the silver nanometer particles is Ag NPs, and the particle size is 25 nm; In step 1, the ultrasonic dispersion conditions are: power 100 w, frequency 40 kHz, time 30 min; In step 1, the concentration of the silver nanometer suspension is 40 mg / L.

2. The seed treatment method according to claim 1, wherein In step 2, the disinfection is carried out with a 5% sodium hypochlorite solution, and the disinfection time is 10 min.

3. The seed treatment method according to claim 1, wherein In step 3, the conditions of the constant temperature shaking incubator are set as: light avoidance, temperature 25°C, rotation speed 100 rpm, and the soaking culture time is 10 h.

4. The seed treatment method according to claim 1, characterized in that In step 4, the specific steps of the paper germination method are: Place the filter paper in a culture dish, add 5 ml of deionized water, and put 15 pumpkin seeds in each culture dish.

5. The seed treatment method according to claim 1, characterized in that, In step 4, the culture conditions of the artificial climate incubator used in the seed treatment method for improving the drought tolerance of pumpkins are: temperature 25°C, humidity 70%, time 5 d, light avoidance culture; the culture conditions of the artificial climate incubator used in the seed treatment method for improving the cold tolerance of pumpkins are: temperature 15°C, humidity 70%, time 15 d, light avoidance culture.

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