Nutrient solution for promoting potato seedling growth and enhancing stress resistance, culture method and application thereof

Abstract

The present invention discloses a nutrient solution, culture method, and application thereof for promoting strong potato seedlings and enhancing stress resistance. Through comparative nutrient solution culture experiments, the present invention discovered an improved 1 / 2 Hoagland nutrient solution. Increasing the amount of phosphorus and zinc promotes short-term strong potato seedlings. Adding seaweed oligosaccharides further promotes the growth of tissue culture seedlings and enhances their stress resistance. Furthermore, the method requires only an ionic liquid nutrient solution for culture, making it suitable for hydroponics and substrate culture. The method is simple, rapid, and effective in promoting strong potato seedlings.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to a nutrient solution for promoting potato seedling growth and enhancing stress resistance, a culture method and an application thereof. Background Art

[0002] The potato is the third largest grain crop in the world and the fourth largest in my country. It is not only an important staple food crop and a basic vegetable crop, but also an excellent feed crop and an industrial raw material crop with enormous value-added potential. Because potatoes are propagated vegetatively, they are primarily grown in tubers. Multi-generational cultivation of tubers can easily lead to the accumulation of various pathogens, such as viruses, viroids, bacteria, and fungi, in the tubers. This prevents the potato plants from fully realizing the variety's production characteristics during production, resulting in a decline in potato yield and quality. To overcome these problems and prevent the "degeneration" of diseased seed potatoes, virus-free seed potatoes are currently widely used to produce virus-free seed potatoes for potato production, increasing yield and improving quality. However, both potato virus-free and factory-scale production of virus-free seed potatoes require the cultivation of tissue culture seedlings.

[0003] Currently, there are two main methods for propagating virus-free potato seedlings in vitro: one is to propagate the seedlings directly in a matrix such as vermiculite, perlite, sand, sawdust, or soil. This method takes a long time to propagate, has a low survival rate, is easily infected with pathogens, and results in high production costs for the original seedlings; the other is to propagate the seedlings in a medium based on MS. This method has a relatively fast propagation speed and a relatively high survival rate, but it requires a high production environment and is expensive to cultivate, also resulting in high production costs for the original seedlings. Therefore, there is an urgent need to develop a culture method and culture medium suitable for culturing potato tissue culture seedlings. Summary of the Invention

[0004] The purpose of the present invention is to overcome the shortcomings and deficiencies of the prior art and provide a nutrient solution that promotes the growth of potato seedlings and enhances stress resistance.

[0005] Another object of the present invention is to provide a cultivation method for promoting the growth of potato seedlings and enhancing stress resistance.

[0006] Another object of the present invention is to provide an application of the above nutrient solution.

[0007] The purpose of the present invention is achieved through the following technical solutions:

[0008] A nutrient solution for promoting potato seedling growth and enhancing stress resistance, comprising: 4.0-6.0 mM KNO3, 2.0-3.0 mM Ca(NO3)2, 2.0-3.0 mM NH4Cl, 0.5-1.5 mM MgSO4, 0.5-1.5 mM KH2PO4, 15.0-25.0 μM H3BO3, 4.0-5.0 μM MnCl2, 1.0-1.4 μM ZnSO4, 0.1-0.2 μM CuSO4, 0.5-1.5 mM K2SO4, 80-100 μM FeSO4, and 0.1-0.2 μM (NH4)6Mo7O 24 , 0.05~0.10mM Fe-Na-EDTA.

[0009] The components of the nutrient solution also include 0.033-0.400 g / L of seaweed oligosaccharide.

[0010] Preferably, the concentration of ZnSO4 in the components of the nutrient solution is 1.2 μM.

[0011] Preferably, the concentration of KH2PO4 in the components of the nutrient solution is 1.0 mM.

[0012] The nutrient solution is used in the cultivation of potato tissue culture seedlings, can promote the rapid growth of the potato tissue culture seedlings and improve their stress resistance.

[0013] Application of the above nutrient solution in potato cultivation.

[0014] The potatoes are potato tissue culture seedlings.

[0015] Application of the above nutrient solution in enhancing potato stress resistance.

[0016] A method for cultivating strong potato tissue culture seedlings comprises the following steps:

[0017] Transplant the potato tissue culture seedlings into the growth medium and add nutrient solution for cultivation.

[0018] The potato tissue culture seedlings are tissue culture seedlings obtained by virus-free culture of potato tissues.

[0019] The growth medium is a nutrient solution or a peat soil matrix.

[0020] When the growth medium is peat soil matrix, the amount of nutrient solution added is twice a week, 100 mL each time.

[0021] The culture time is 20 to 60 days.

[0022] The culture temperature is 20-22°C.

[0023] The light intensity of the culture is 5 to 15 Klux.

[0024] The lighting time of the culture is 12 to 16 hours per day.

[0025] The culture medium is 1 / 2 Hoagland nutrient solution; preferably 1 / 2 Hoagland nutrient solution added with seaweed oligosaccharide.

[0026] The present invention has the following advantages and effects compared to the prior art:

[0027] Due to the limited space in the growth medium and tissue culture flask, potato tissue culture seedlings cannot grow up to the requirements of being transplanted to potted plants or fields. In order to promote the rapid growth and robustness of potato tissue culture seedlings in a short period of time, the common method is to change the culture medium composition. However, due to the limitations of the tissue culture device and environmental differences, it is still impossible to expand robust potato seedlings. Recently, studies have found that potato seedlings can be promoted to grow by adding nutrient solution to vermiculite. However, this method has complex nutrient solution components and a cumbersome planting process, which is not ideal for promoting the growth of potato tissue culture seedlings. For this reason, the present invention has found a modified 1 / 2 Hoagland nutrient solution through a comparative test of nutrient solution culture. Increasing the amount of phosphorus and zinc elements can promote the short-term robustness of potato seedlings. Adding seaweed oligosaccharides can better promote the growth of tissue culture seedlings and enhance their stress resistance. Moreover, only ionic liquid nutrient solution needs to be used for culture, which is suitable for water culture and matrix culture. The method is simple and fast, and has a good promoting effect. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 These are potato plants used in the hydroponic environment and culture test in the examples.

[0029] Figure 2 This is a graph showing the plant yield results of the culture experiments with different culture solutions in Example 1.

[0030] Figure 3 This is a graph showing the dry weight results of plants grown in the culture experiments using different culture solutions in Example 1.

[0031] Figure 4 This is a graph showing the results of the culture experiment in Example 3 in which the phosphorus concentration was adjusted alone.

[0032] Figure 5 This is a graph showing the results of the culture experiment in Example 3 in which the zinc concentration was adjusted alone.

[0033] Figure 6 This is a graph showing the results of a culture test in Example 4 in which different concentrations of seaweed oligosaccharides were added to the improved culture medium.

[0034] Figure 7 1 is a graph showing the results of the culture experiment at different light intensities in Example 5.

[0035] Figure 8This is a graph showing the results of detecting the expression levels of related enzymes in plants cultured in different improved culture solutions after low temperature treatment in Example 6.

[0036] Figure 9 This is a graph showing the results of detecting the expression levels of related enzymes in plants cultured in different improved culture solutions after high temperature treatment in Example 6.

[0037] Figure 10 This is a graph showing the expression level detection results of related genes in plants cultured in different improved culture solutions after low temperature treatment in Example 6; positive values ​​represent the results of each treatment compared with treatment T9, and negative values ​​represent the expression results of treatment T9 compared with other treatments.

[0038] Figure 11 This is a graph showing the expression level detection results of related genes in plants cultured in different improved culture solutions after high temperature treatment in Example 6; positive values ​​represent the results of each treatment compared with treatment T9, and negative values ​​represent the expression results of treatment T9 compared with other treatments. DETAILED DESCRIPTION

[0039] The present invention will be described in further detail below with reference to the embodiments and drawings, but the embodiments of the present invention are not limited thereto.

[0040] If specific experimental conditions are not specified in the following embodiments, conventional experimental conditions or those recommended by the reagent company will generally be used. Materials and reagents used were commercially available unless otherwise specified.

[0041] Example 1 Screening of basal culture fluid

[0042] 1.1 Cultivation of potato tissue culture seedlings

[0043] Prepare the culture medium with 4.43g / L MS powder, 30g / L sucrose, 15g / L agar, and pH 5.8-6.0, then sterilize at 121℃ for 20min and dispense into tissue culture bottles. Transfer the single-node potato stem segment with axillary buds into the tissue culture bottle containing the culture medium and place it in a light culture bottle at a temperature of 20-22℃ and a light intensity of 8-10Klux for 14 hours. After 4-6 weeks, observe that the height of the tissue culture seedlings is about 2 / 3 of the height of the tissue culture bottle and can be used for transplanting. 1.2 Basic culture medium seedling cultivation experiment

[0044] The potato tissue culture seedlings obtained in 1.1 were transplanted into an aeroponic chamber for hydroponic culture. The potato plants were cultured using four different nutrient solutions: 1 / 2 Hoagland, MS, GSM, and PD. The culture temperature was 22°C, the light intensity was 8-10 Klux, and the photoperiod was 16 hours. After 60 days of culture, the whole plant was collected, the mass of the tubers was measured to calculate the yield, and the plants were weighed to calculate the dry weight.

[0045] Treatment 1: 1 / 2 Hoagland medium, whose composition is 5.0mM KNO3, 2.5mM Ca(NO3)2, 2.5mM NH4Cl, 1.0mM MgSO4, 0.5mM KH2PO4, 20μM H3BO3, 4.5μM MnCl2, 1.0μM ZnSO4, 0.16μM CuSO4, 1.0mM K2SO4, 90μM FeSO4, 0.16μM (NH4)6Mo7O 24 and 0.08 mM Fe-Na-EDTA;

[0046] Treatment 2: MS culture medium, whose composition is 37.5mM KNO3, 4.0mM Ca(NO3)2, 20.6mM NH4Cl, 1.7mMMgSO4, 1.25mM KH2PO4, 100μM H3BO3, 100μM MnCl2, 25μM ZnSO4, 0.25μM CuSO4, 0.01mM K2SO4, 1.0μM (NH4)6Mo7O 24 and 0.08 mM Fe-Na-EDTA;

[0047] Treatment 3: GSM culture medium, whose composition is 28.5mM KNO3, 0.8mM Ca(NO3)2, 2.5mM NH4Cl, 1.2mMMgSO4, 1.25mM KH2PO4, 84μM H3BO3, 10μM MnCl2, 3.0μM ZnSO4, 2.6μM CuSO4, 0.07mM K2SO4, 90μM FeSO4, 2.8μM (NH4)6Mo7O 24 and 0.08 mM Fe-Na-EDTA;

[0048] Treatment 4: PD culture medium, whose composition is 15mM KNO3, 2.5mM Ca(NO3)2, 3.0mM NH4Cl, 1.5mMMgSO4, 1.0mM KH2PO4, 60μM H3BO3, 9.0μM MnCl2, 2.0μM ZnSO4, 0.8μM CuSO4, 0.2mM K2SO4, 45μM FeSO4, 1.0μM (NH4)6Mo7O 24 and 0.08 mM Fe-Na-EDTA;

[0049] The experimental results are as follows Figure 2 and Figure 3As shown, treatment 1 had the highest potato yield, with an average of 278g of tubers per plant, followed by treatment 2 at 216g. Treatment 4 had the lowest yield, with tubers weighing 16g per plant. Treatment 1 had the highest dry weight, with an average of 34.2g per plant, followed by treatment 4 at 26.7g per plant. Treatment 2 had the lowest dry weight, at 18.6g per plant. This demonstrated that 1 / 2 Hoagland medium was the most effective for strengthening seedlings, and subsequent experiments used 1 / 2 Hoagland medium as the basal medium.

[0050] Example 2 Screening of Growth Medium

[0051] 2.1 Experiment on seedling cultivation in different growth media

[0052] Potato tissue culture seedlings were obtained by culturing according to the method of 1.1 in Example 1, cultured in 1 / 2 Hoagland culture medium, transplanted to different growth media for treatment and culture, and collected and tested after 30 days of culture under natural light.

[0053] Treatment 1: agar + 1 / 2 Hoagland medium, tissue culture flask growth;

[0054] Treatment 2: nutrient-free peat soil substrate + 1 / 2 Hoagland nutrient solution, supplemented with nutrient solution twice a week, 100 mL each time;

[0055] Treatment 3: absorbent cotton + 1 / 2 Hoagland nutrient solution, use absorbent cotton to fix the tissue culture seedlings in the hydroponic bread box, and culture the tissue culture seedlings in liquid form.

[0056] The experimental results are shown in Table 1. After 30 days of growth under the same growth environment, treatment 3 showed significant advantages in root length, root number, plant height, internode number, leaf number, and plant dry weight. Compared with treatment 1, treatment 3 significantly increased root length, root number, plant height, internode number, leaf number, and plant dry weight by 35.2%, 57.1%, 47.2%, 29.2%, 61.4%, and 74.4%, respectively. This indicates that treatment 3 is more suitable for the growth of potato tissue culture seedlings and produces the strongest seedlings. However, treatment 3 does not simulate the actual field growth environment. The temperature buffering effect of the soil matrix on the growth of potato seedlings is different from that in a culture medium. In addition, hydroponics requires complex equipment such as aerosol chambers, which is complex and costly to operate, making it unsuitable for large-scale production and application. To more comprehensively verify the experimental results, subsequent experiments used hydroponics and soil matrix as the growth medium to verify their seedling-strengthening effects.

[0057] Table 1 Results of seedling cultivation experiments in different growth media

[0058] deal with Root length / cm Number of roots Plant height / cm Number of internodes Robustness Number of blades Plant dry weight / g Process 1 16.5 4.2 10.6 4.8 Poor 12.7 1.25 Process 2 18.4 5.9 12.4 5.4 Stronger 16.9 1.59 Process 3 22.3 6.6 15.6 6.2 robust 20.5 2.18

[0059] 2.2 Effects of different nutrient solution formulas on potato growth in matrix tissue culture seedling cultivation

[0060] Potato tissue culture seedlings were obtained by culturing according to the method of 1.1 in Example 1, transplanted into pots with peat soil matrix as the growth medium, and watered with different nutrient solutions for culture. The nutrient solution was supplemented twice a week, 100 mL each time, and the same amount of water was supplemented according to growth needs. The same management was applied to each treatment, natural light was used, and the seedlings were collected and tested after 40 days of culture.

[0061] Treatment 1: substrate + commercially available compound fertilizer (15-15-15) at a conventional dosage;

[0062] Treatment 2: substrate + 1 / 2 Hoagland nutrient solution;

[0063] Treatment 3: substrate + MS nutrient solution;

[0064] Treatment 4: substrate + GSM nutrient solution;

[0065] Treatment 5: substrate + PD nutrient solution.

[0066] The experimental results are shown in Table 2. Comparing the various treatments, it was found that Treatment 2 had the longest root length, plant height, number of leaves and plant dry weight, and Treatment 2 had the best robustness, indicating that the nutrient solution scheme of Treatment 2 was better than the traditional compound fertilizer scheme, and among the four nutrient solution schemes, the nutrient solution scheme of Treatment 2 was the best.

[0067] Table 2 Effects of different nutrient solution formulas on potato growth in matrix tissue culture seedling cultivation

[0068]

[0069] Example 3 Effect of different phosphorus / zinc dosages on the growth of potato tissue culture seedlings

[0070] 3.1 Experiment on seedling cultivation by adjusting phosphorus and zinc dosage separately

[0071] Potato tissue culture seedlings were obtained by culturing according to the method of 1.1 in Example 1, transplanted into an aeroponic chamber, and cultured in 1 / 2 Hoagland culture medium containing different concentrations of phosphorus and zinc under natural light. After 30 days of culture, the seedlings were collected and tested.

[0072] Phosphorus treatment: only the phosphorus concentration of 1 / 2 Hoagland nutrient solution was changed, while the concentrations of other elements remained unchanged. The phosphorus concentrations were 0.5, 1.0, and 1.5 mM respectively;

[0073] Zinc treatment: Only the zinc concentration was changed in 1 / 2 Hoagland nutrient solution, while the concentrations of other elements remained unchanged. The zinc concentrations were 1.0, 1.2, and 1.4 μM, respectively.

[0074] The experimental results are as follows Figure 4 and Figure 5 As shown, different phosphorus concentration treatments significantly affected plant height, branch number, fresh weight, and dry weight of potato tissue culture seedlings. Compared with P0.5, plant height and fresh weight increased significantly in P1.0 and P1.5, but there were no significant differences between P1.0 and P1.5. Plant dry weight measurements revealed a significant increase in P1.0, reaching a maximum of 1.74 g, followed by P1.5, indicating that phosphorus concentration P1.0 is more suitable for the growth of potato tissue culture seedlings. Similarly, compared with Zn1.0, Zn1.2 and Zn1.4 significantly increased plant fresh weight and dry weight, respectively, but there were no significant differences in plant height and root length. There were no significant differences between Zn1.2 and Zn1.4 in any of the measured parameters, indicating that zinc concentration Zn1.2 is more suitable for the cultivation of potato tissue culture seedlings.

[0075] 3.2 Simultaneous adjustment of phosphorus and zinc dosage to strengthen seedling cultivation experiment

[0076] Potato tissue culture seedlings were obtained by culturing according to the method of 1.1 in Example 1, transplanted into a hydroponic box, and cultured in 1 / 2 Hoagland culture medium containing different concentrations of phosphorus and zinc under natural light. After culturing for 25 days, the seedlings were collected and tested.

[0077] Treatment 1: 1 / 2 Hoagland nutrient solution;

[0078] Treatment 2: zinc + 1 / 2 Hoagland nutrient solution was added, and the zinc treatment was changed from 1.0 μM ZnSO4 in treatment 1 to 1.2 μM ZnSO4;

[0079] Treatment 3: phosphorus + 1 / 2 Hoagland nutrient solution was added, and the phosphorus treatment was changed from 0.5mM KH2PO4 in treatment 1 to 1.0mM KH2PO4;

[0080] Treatment 4: Zinc and phosphorus + 1 / 2 Hoagland nutrient solution were added at the same time. The zinc treatment was changed from 1.0μM ZnSO4 in treatment 1 to 1.2μM ZnSO4; the phosphorus treatment was changed from 0.5mM KH2PO4 in treatment 1 to 1.0mM KH2PO4.

[0081] The experimental results are shown in Table 3. After 25 days under the same growth conditions, the modified nutrient solution in Treatments 2, 3, and 4 significantly increased root length, lateral root number, plant height, stem diameter, leaf number, and plant dry weight. Compared with Treatment 1, stem diameter, leaf number, and dry weight increased by 3.3%, 4.9%, 17.9%, 2.8%, 6.2%, 13.5%, 5.2%, 15.6%, and 30.7%, respectively, in Treatments 2, 3, and 4, respectively. The data indicate that increasing zinc or phosphorus concentration in Treatments 2 and 3 promoted growth in potato stem diameter, leaf number, and plant biomass. Simultaneously increasing both zinc and phosphorus concentrations in Treatment 4 significantly enhanced the growth of potato tissue culture seedlings, indicating that zinc and phosphorus interact to promote tissue culture seedling growth. Therefore, the following experiments were further conducted using modified zinc and phosphorus concentrations.

[0082] Table 3 Simultaneous adjustment of phosphorus and zinc dosages for seedling cultivation

[0083]

[0084] 3.3 Interaction between phosphorus and zinc in promoting the growth of potato tissue culture seedlings in substrate cultivation

[0085] Potato tissue culture seedlings were obtained by culturing according to the method in 1.1 of Example 1, transplanted into a matrix growth medium, and cultured with different nutrient solutions under natural light. After culturing for 30 days, the seedlings were collected and tested.

[0086] Treatment 1: substrate + water;

[0087] Treatment 2: substrate + 1 / 2 Hoagland nutrient solution;

[0088] Treatment 3: substrate + zinc + 1 / 2 Hoagland nutrient solution, the zinc treatment was changed from 1.0 μM ZnSO4 in treatment 1 to 1.2 μM ZnSO4;

[0089] Treatment 4: substrate + phosphorus + 1 / 2 Hoagland nutrient solution, the phosphorus treatment was changed from 0.5 mM KH2PO4 in treatment 1 to 1.0 mM KH2PO4 (same as treatment 3 in 3.2);

[0090] Treatment 5: substrate + zinc + phosphorus + 1 / 2 Hoagland nutrient solution, the zinc treatment was changed from 1.0 μM ZnSO4 in treatment 1 to 1.2 μM ZnSO4; the phosphorus treatment was changed from 0.5 mM KH2PO4 in treatment 1 to 1.0 mM KH2PO4 (same as treatment 4 in 3.2).

[0091] The experimental results are shown in Table 4. Compared with Treatment 1, nutrient solution treatments 2, 3, 4, and 5 significantly increased root length, lateral root number, plant height, stem diameter, leaf number, and plant dry weight after 30 days under the same growth conditions. Observation of stem diameter, leaf number, and dry weight revealed increases of 6.0%, 0.6%, 20.6%, 10.0%, 13.3%, 42.2%, 28.0%, 15.2%, and 68.6%, respectively, compared with Treatment 2. Analysis of the data revealed that increasing zinc or phosphorus concentration in Treatments 3 and 4 promoted stem diameter, leaf number, and plant biomass. Simultaneously increasing both zinc and phosphorus concentrations in Treatment 5 significantly enhanced the growth of potato tissue culture seedlings, indicating that zinc and phosphorus interact to promote tissue culture seedling growth, consistent with the results of hydroponic studies.

[0092] Table 4 Substrate culture + changing the amount of phosphorus and zinc to promote the growth of potato tissue culture seedlings

[0093]

[0094] Example 4 Effect of adding different dilution multiples of seaweed oligosaccharides to improve nutrient solution on the growth of potato seedlings

[0095] 4.1 Adding seaweed oligosaccharides at different dilution ratios under hydroponic conditions

[0096] Potato tissue culture seedlings were obtained by culturing according to the method of 1.1 in Example 1, transplanted into a hydroponic box, cultured in different nutrient solutions, and seaweed oligosaccharides were added to the experimental group (the product name is brown algae oligosaccharide, the specification is 99%, purchased from Xi'an Yatu Biotechnology Co., Ltd., the seaweed oligosaccharide powder was prepared into a 20 g / L stock solution, and the final concentration was diluted 50 to 600 times and added to the nutrient solution), exposed to natural light, and collected and tested after culturing for 22 days.

[0097] Treatment 1: Normal culture in hydroponics with deionized water;

[0098] Treatment 2: Normal culture in hydroponic culture using 1 / 2 Hoagland-i nutrient solution (same as Treatment 4 in 3.2 of Example 3);

[0099] Treatment 3: nutrient solution 1 / 2 Hoagland-i + 0.400 g / L seaweed oligosaccharide;

[0100] Treatment 4: nutrient solution 1 / 2 Hoagland-i + 0.200 g / L seaweed oligosaccharide;

[0101] Treatment 5: nutrient solution 1 / 2 Hoagland-i + 0.100 g / L seaweed oligosaccharide;

[0102] Treatment 6: nutrient solution 1 / 2 Hoagland-i + 0.067 g / L seaweed oligosaccharide;

[0103] Treatment 7: nutrient solution 1 / 2 Hoagland-i + 0.050 g / L seaweed oligosaccharide;

[0104] Treatment 8: nutrient solution 1 / 2 Hoagland-i + 0.040 g / L seaweed oligosaccharide;

[0105] Treatment 9: nutrient solution 1 / 2 Hoagland-i + 0.033 g / L seaweed oligosaccharide;

[0106] The experimental results are shown in Table 5. Treatment 4 had the best effect. Compared with treatment 2, the plant height, number of leaves, stem diameter and dry weight increased by 27.27%, 41.75%, 21.11% and 93.65%, respectively, indicating that treatment 4 with the addition of 0.200 g / L seaweed oligosaccharides was more suitable for the growth of potato seedlings.

[0107] Table 5 Effects of different dilution ratios of seaweed oligosaccharides and improved nutrient solution on the growth of potato seedlings

[0108]

[0109] Note: The growth rate is compared with treatment 2

[0110] 4.2 Effects of different dilution ratios of seaweed oligosaccharide treatment and modified nutrient solution on the growth of potato seedlings in substrate cultivation

[0111] Potato tissue culture seedlings were obtained by culturing according to the method of 1.1 in Example 1, transplanted into an aeroponic chamber, using a substrate as a growth medium, and poured with different nutrient solutions for cultivation under natural light. After culturing for 26 days, the seedlings were collected and tested.

[0112] Treatment 1: normal culture in hydroponics with substrate + deionized water;

[0113] Treatment 2: Normal culture in hydroponics using substrate and nutrient solution 1 / 2 Hoagland-i (modified to 1.2 μM ZnSO4 and 1.0 mM KH2PO4 after experiment);

[0114] Treatment 3: substrate + nutrient solution 1 / 2 Hoagland-i + 0.400 g / L seaweed oligosaccharide;

[0115] Treatment 4: substrate + nutrient solution 1 / 2 Hoagland-i + 0.200 g / L seaweed oligosaccharide;

[0116] Treatment 5: substrate + nutrient solution 1 / 2 Hoagland-i + 0.100 g / L seaweed oligosaccharide;

[0117] Treatment 6: substrate + nutrient solution 1 / 2 Hoagland-i + 0.067 g / L seaweed oligosaccharide;

[0118] Treatment 7: substrate + nutrient solution 1 / 2 Hoagland-i + 0.050 g / L seaweed oligosaccharide;

[0119] Treatment 8: substrate + nutrient solution 1 / 2 Hoagland-i + 0.040 g / L seaweed oligosaccharide;

[0120] Treatment 9: substrate + nutrient solution 1 / 2 Hoagland-i + 0.033 g / L seaweed oligosaccharide;

[0121] The experimental results are as follows Figure 6 As shown, treatment 4 had the best effect. Compared with treatment 2, plant height, leaf number, stem diameter and dry weight increased by 20.0%, 28.2%, 24.3% and 98.65%, respectively. The differences in stem diameter and dry weight were significant, indicating that treatment 4 with the addition of 0.200 g / L seaweed oligosaccharides was also suitable for the growth of potato seedlings in substrate cultivation.

[0122] Example 5 Effects of different light intensities on the growth of potatoes cultured in nutrient solution supplemented with seaweed oligosaccharides

[0123] 5.1 Cultivation experiment under different light intensities under hydroponic conditions

[0124] Potato tissue culture seedlings were obtained by culturing according to the method of 1.1 in Example 1, transplanted into hydroponic conditions, and cultured using the culture medium of Treatment 4 in Example 4. Different light intensities were set and the seedlings were collected and tested after culturing for 18 days.

[0125] Light treatment L1: light intensity from the light source to the top of the plant growth is 5Klux;

[0126] Light treatment L2: light intensity from the light source to the top of the plant growth is 10Klux;

[0127] Light treatment L3: light intensity from the light source to the top of the plant growth is 15Klux;

[0128] The experimental results are as follows Figure 7 As shown in the figure, different light intensity treatments significantly affected the plant height, stem diameter, and dry weight of potato tissue culture seedlings. Compared with L1, the plant height and stem diameter of L2 and L3 increased significantly, but there was no significant difference in plant height between L2 and L3. The dry weight of the plants was significantly increased in the L2 treatment, with the dry weight of the L2 treatment increasing by 96.7% and 35.9% compared with the fresh weight of the L1 and L3 treatments, respectively. This indicates that light intensity L2 is more suitable for the growth of potato tissue culture seedlings.

[0129] 5.2 Culture experiments under different light intensities under matrix culture conditions

[0130] Potato tissue culture seedlings were obtained by culturing according to the method in 1.1 of Example 1, transplanted into a matrix growth medium, and poured with the culture solution of Treatment 4 in Example 4. Different light intensities were set, and the seedlings were collected and tested after culturing for 23 days.

[0131] Light treatment L1: light intensity from the light source to the top of the plant growth is 5Klux;

[0132] Light treatment L2: light intensity from the light source to the top of the plant growth is 10Klux;

[0133] Light treatment L3: light intensity from the light source to the top of the plant growth is 15Klux;

[0134] The experimental results are shown in Table 6. Different light intensity treatments significantly affected the plant height, stem diameter, and dry weight of potato tissue culture seedlings. Compared with L1, plant height and stem diameter increased significantly in L2 and L3, with growth rates of 38.26% and 146.03% and 15.25% and 119.86%, respectively. Plant dry weight measurements revealed a significant increase in potato dry weight in the L2 treatment, with fresh weight increases of 103.2% and 13.4% in the L2 treatment compared to treatments L1 and L3, respectively. This indicates that light intensity L2 is more suitable for the growth of potato tissue culture seedlings in substrate cultivation.

[0135] Table 6 Effects of different light intensities on the improvement of nutritional culture tissue culture seedlings by adding seaweed oligosaccharides

[0136]

[0137] Example 6 Comprehensive evaluation of phosphorus and zinc nutrition and seaweed oligosaccharides on high and low temperature resistance

[0138] According to the results of the above experiments, it can be seen that adjusting the content of phosphorus / zinc and seaweed oligosaccharides in the nutrient solution has a significant effect on the effect of potato seedling growth. In order to further study the effects of different culture medium combinations on potato seedlings, this experiment conducted experimental tests on the stress resistance of potato seedlings.

[0139] Potato tissue culture seedlings were obtained by culturing according to the method of 1.1 in Example 1, and transplanted into a light incubator with a light intensity of 10 Klux at the top of the plant growth area. The substrate was used as a growth medium and different nutrient solutions were poured into the culture. After 28 days of culture, a control group, a low temperature treatment group, and a high temperature treatment group were set up for each treatment. After the treatment, the potato plant leaves were collected and stored at -80°C for detection of leaf enzyme activity and purple acid phosphatase-related gene expression. Three replicates were set for each group.

[0140] Treatment T1: substrate + nutrient solution 1 / 2 Hoagland-i (Zn1.2+P1.0) + 0.200 g / L seaweed oligosaccharide;

[0141] Treatment T2: substrate + nutrient solution 1 / 2 Hoagland-i (Zn1.2+P0.5) + 0.200 g / L seaweed oligosaccharide;

[0142] Treatment T3: substrate + nutrient solution 1 / 2 Hoagland-i (Zn1.2+P1.5) + 0.200 g / L seaweed oligosaccharide;

[0143] Treatment T4: substrate + nutrient solution 1 / 2 Hoagland-i (Zn1.0+P0.5) + 0.200 g / L seaweed oligosaccharide;

[0144] Treatment T5: substrate + nutrient solution 1 / 2 Hoagland-i (Zn1.4+P0.5) + 0.200 g / L seaweed oligosaccharide;

[0145] Treatment T6: substrate + nutrient solution 1 / 2 Hoagland-i (Zn1.2+P1.0) + 0.400 g / L seaweed oligosaccharide;

[0146] Treatment T7: substrate + nutrient solution 1 / 2 Hoagland-i (Zn1.2+P1.0) + 0.050 g / L seaweed oligosaccharide;

[0147] Treatment T8: substrate + 0.200 g / L seaweed oligosaccharide;

[0148] Treatment T9: substrate + nutrient solution 1 / 2 Hoagland-i (Zn1.2+P1.0).

[0149] The concentration of Zn in the above nutrient solutions is in μmol, the concentration of P is in mmol, and the concentration of other nutrients refers to the nutrient solution 1 / 2 Hoagland.

[0150] Low temperature treatment: The treatment temperature was -2°C. Leaf samples were collected 5 hours after treatment for enzyme activity analysis (Solabo kit) and gene expression level detection.

[0151] High temperature treatment: The treatment temperature was 38°C. After 5 hours of treatment, leaf samples were collected for enzyme activity analysis (Solabo kit) and gene expression detection.

[0152] Enzyme activity detection: The analysis method of enzyme activity POD, CAT, MDA, SOD and Pro was determined according to the operation method of Solebol kit ( Science & Technology Co. Beijing, China).

[0153] Gene expression analysis: In order to more comprehensively evaluate the resistance changes of potatoes after seedling strengthening treatment with different culture media, the expression level of purple acid phosphatase (PAP), a related gene that plays an important role in the stress resistance process in the collected samples, was analyzed. The PAP gene is an important gene that responds to phosphorus starvation and activates the utilization of organic phosphorus sources inside and outside the plant body, and is involved in regulating stress and hormone changes. According to the potato genome information website the Spud DB Potato Genomics Resources (DM v6.1, http: / / spuddb.uga.edu) (Rudic, J., Dragicevic, MB, Momcilovic, I., Simonovic, AD, Pantelic, D., 2022. In Silico Study of Superoxide Dismutase Gene Family in Potato and Effects of Elevated Temperature and Salicylic Acidon Gene Expression. Antioxidants 11(3), 488.), the key purple acid phosphatase (PAP)-related genes retrieved are Soltu.DM.01G049890, Soltu.DM.01G049900, Soltu.DM.03G019440, Soltu.DM.03G022790, Soltu.DM.04G003760 and Soltu.DM.04G036010. Primers were designed for the six PAP genes identified using the bioinformatics software PrimerPremier 6.0, and gene expression was measured by quantitative real-time PCR (qRT-PCR). RNA extraction and reverse transcription were performed from plant leaves according to the soybean RNA extraction method (Chengchen, Li, Shunhua, Gui, Tao, & Yang, et al. (2011). Identification of Soybean Purple Acid Phosphatase Genes and Their Expression Responses to Phosphorus Availability and Symbiosis. International Symposium on Integrative Plant Biology).All samples were analyzed on a CFX96 Real-Time PCR Detection System (Bio-Rad, Hercules, USA). The potato housekeeping gene EF1A (F: 5'-227ATTGGAAACGGATATGCTCCA-3'; R: 5'-TCCTTACCTGAACGCCTGTCA-3') was used as an endogenous control for normalization. qRT-PCR was performed using a 20 μL reaction mixture consisting of 2 μL cDNA template, 0.8 μL forward primer, 0.8 μL reverse primer, 10 μL 2× SYBR qPCR Master mix, and 6.4 μL RNase-free H2O. Thermal cycling conditions were as follows: initial denaturation at 95°C for 2 minutes, followed by denaturation at 95°C for 15 seconds, and fluorescence signal collection at 60°C for 30 seconds, repeated 40 times. Each experimental treatment included four biological replicates, and gene expression levels were calculated using a 2. -ΔCt Method calculation (Wang, Y., Li, Y., Zhou, F., Zhang, L., Gong, J., Cheng, C. et al., 2023. Genome-wide characterization, phylogenetic and expression analysis of Histone gene family in cucumber (Cucumis sativus L.). Int. J. Biol. Macromol. 230, 123401.).

[0154] The experimental results are shown in Table 7 and Figures 8-11 The data in Table 7 show that different nutrient solutions and seaweed oligosaccharide treatments affected the plant height, stem diameter, and dry weight of potato tissue culture seedlings. In contrast, treatment 1 had the largest data for stem diameter, leaf area, biomass, and root-to-shoot ratio, significantly promoting the growth of potato tissue culture seedlings.

[0155] Table 7 Effects of different nutrient solution concentrations and seaweed oligosaccharides on potato growth

[0156]

[0157] like Figure 8 As shown in the figure, the enzyme activities of different nutrient solutions and seaweed oligosaccharide treatments changed differently after 5 hours of low temperature treatment. As can be seen from the figure, treatment 1 has higher POD, CAT, MDA, SOD and Pro enzyme activities, indicating that the treatment nutrient solution combination has improved the low temperature resistance compared with other treatments.

[0158] like Figure 9As shown in the figure, the enzyme activities of different nutrient solutions and seaweed oligosaccharide treatments changed differently after 5 hours of high temperature treatment. As can be seen from the figure, treatment 1 has higher POD, CAT, MDA, SOD and Pro enzyme activities, indicating that the nutrient solution combination of treatment 1 has improved the heat resistance compared with other treatments.

[0159] Figure 10 qRT-PCR analysis of the expression levels of potato PAP-related genes showed that after treatment at -2℃ for 5 hours, compared with the control treatment T9, under the same phosphorus and zinc nutrients, the addition of different concentrations of seaweed oligosaccharides to treatments T1, T6 and T7, the genes Soltu.DM.01G049890 and Soltu.DM.01G049900 showed the largest negative increase in gene expression in the leaves of T1 treatment, indicating that when seaweed oligosaccharides were added to treatments T1, T6 and T7, the PAP expression levels were significantly reduced compared with the control treatment T9 without adding seaweed oligosaccharides, and the reduction in treatment T1 was the largest, revealing that under the T1 treatment, potato plants obtained the least utilization of organic phosphorus in the leaves while meeting the growth and obtaining maximum biomass, while compared with treatment T1, the demand and utilization of organic phosphorus in treatments T6 and T7 increased. Similarly, the expression of the genes in Soltu.DM.03G019440 and Soltu.DM.03G022790 increased the least in the T1 treatment, while the expression of genes in T6 and T7 increased more significantly compared to the T1 treatment, indicating that the expression of genes involved in organic phosphorus utilization was lower in the T1 treatment. The consistent expression of these four genes indicates that the addition of seaweed oligosaccharides to the T1 treatment increased the availability of phosphorus nutrients under chilling stress, enhancing the potato plants' resistance to chilling stress.

[0160] Figure 11qRT-PCR analysis of potato PAP-related gene expression revealed that after 5 hours of treatment at 38°C, compared to the control treatment T9, under the same phosphorus and zinc nutrients, the expression of genes Soltu.DM.01G049890, Soltu.DM.01G049900, and Soltu.DM.04G036010 increased the most in T1 treatment, with the addition of different concentrations of seaweed oligosaccharides compared to the control treatment T9. This suggests that seaweed oligosaccharides may be involved in regulating potato phosphorus nutrition. The expression level of Soltu.DM.01G049890 was significantly lower than that of the control treatment T9 without seaweed oligosaccharides, with the largest decrease in T1 treatment. This suggests that under T1 treatment, potato plants utilize the least organic phosphorus to achieve maximum biomass while meeting growth targets, while the demand for organic phosphorus in treatments T6 and T7 increases compared to T1. Similarly, Soltu.DM.01G049900 and Soltu.DM.04G036010 showed the greatest fold decrease in expression in the T1 treatment, while T6 and T7 showed a greater fold increase in expression compared to T1, indicating that under the T1 nutrient combination, the expression of PAP genes, which are involved in the acquisition and utilization of organic phosphorus by potato plants, was lower. Similarly, the expression of Soltu.DM.04G022790 increased the least in T1 compared to T9. Overall, the expression of these four genes consistently indicates that under heat stress, the addition of seaweed oligosaccharides in T1 improved phosphorus availability, allowing plants to more fully obtain and utilize the phosphorus they need from the growth medium, resulting in lower phosphorus starvation signals. This suggests that the T1 nutrient combination is more effective, improving nutrient utilization, achieving optimal biomass, and enhancing heat tolerance.

[0161] In general, different improved nutrient solutions will have a significant impact on the stress resistance of potato tissue culture seedlings. The improved nutrient solution with added seaweed oligosaccharides has better effects than the improved nutrient solution without additions. Among them, the T1 group has the most significant growth promotion effect and the most obvious improvement in stress resistance. After high temperature / low temperature treatment, the expression levels of stress-related enzymes and genes have been significantly improved.

[0162] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be considered as equivalent replacement methods and are included in the scope of protection of the present invention.

Claims

1. A nutrient solution for promoting potato seedling growth and enhancing stress resistance, characterized in that: The components of the nutrient solution include: 4.0-6.0 mM KNO3, 2.0-3.0 mM Ca(NO3)2, 2.0-3.0 mM NH4Cl, 0.5-1.5 mM MgSO4, 0.5-1.5 mM KH2PO4, 15.0-25.0 μM H3BO3, 4.0-5.0 μM MnCl2, 1.0-1.4 μM ZnSO4, 0.1-0.2 μM CuSO4, 0.5-1.5 mM K2SO4, 80-100 μM FeSO4, 0.1-0.2 μM (NH4)6Mo7O 24 ,0.05~0.10 mM Fe-Na-EDTA.

2. The nutrient solution according to claim 1, wherein: The components of the nutrient solution also include 0.033-0.400 g / L of seaweed oligosaccharide.

3. The nutrient solution according to claim 1, wherein: The concentration of ZnSO4 in the components of the nutrient solution is 1.2 μM; The concentration of KH2PO4 in the components of the nutrient solution is 1.0 mM.

4. Use of the nutrient solution according to claim 1 in culturing potatoes.

5. A method for cultivating strong potato seedlings by tissue culture, characterized in that The steps include: Transplanting the potato tissue culture seedlings into a peat soil matrix, adding the nutrient solution according to any one of claims 2 to 3 for cultivation; The amount of nutrient solution added is twice a week, 100 mL each time; The light intensity of the culture is 10-15Klux.

6. The culture method according to claim 5, wherein: The culture time is 20 to 60 days; The culture temperature is 20-22°C.

7. The culture method according to claim 5, wherein: The lighting time of the culture is 12 to 16 hours per day.

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