Method for improving propagation efficiency of potato tissue culture stems

Abstract

The invention provides a method for improving the propagation efficiency of a potato tissue culture stem, which comprises the following steps: inoculating an Atlantic variety sterile stem section as an explant into an MS basal culture medium containing 3.0% of sucrose and 7g / L of agar and having a pH value of 5.8, supplementing an optimized culture solution containing 100mg / L of calcium nitrate and 3.0% of sucrose into a culture container for three times on the 0th day, the 5th day and the 10th day of culture, and culturing for 3-5 days. The whole process is cultured for 20 days under the conditions of 24 DEG C, 16 hours of illumination / 8 hours of darkness and 4000 lux, an optimized culture solution is prepared by loading active ingredients on an alginate microcarrier, carrying out staged temperature control stirring, adding selenized carbon black and sodium succinate under the protection of nitrogen, carrying out shading curing at 25 DEG C, and carrying out sterile filtration, and the terminal dissolved oxygen content is less than or equal to 0.5 ppm. The method remarkably shortens the potato stem culture period, improves the breeding efficiency and is suitable for large-scale production of the detoxified seed potatoes.

Description

Technical Field

[0001] The present application relates to the technical field of potatoes, and in particular to a method for improving the propagation efficiency of potato tissue culture stems. Background Art

[0002] As an important global food and cash crop, the potato's industrial value has long surpassed the simple scope of consumption, profoundly influencing the sustainable development of food processing, bioenergy, and modern agriculture. However, the core pain point of the potato industry—the stable and efficient supply of healthy seed potatoes—has long been constrained by inherent technical bottlenecks in the seed potato propagation process. Among them, the industrialized production of virus-free seed potatoes is particularly critical, as it not only affects the yield potential, but also directly determines the quality grade and disease resistance of commercial potatoes. The current mainstream production model mainly relies on two major systems: greenhouse field propagation and tissue culture technology, but both are mired in a sharp contradiction between efficiency and cost.

[0003] While greenhouse production systems can achieve a certain scale of seed potato production, they face the fatal drawback of repeated pest and disease infestation. Pathogens carried by soil or vectors can easily accumulate over generations, rapidly diminishing the virus-free advantage. Even more troubling, their production capacity is highly dependent on natural climate rhythms. Light cycles and temperature fluctuations cannot be precisely controlled by humans, resulting in long production cycles and significant batch-to-batch quality fluctuations. In contrast, sterile tissue culture-based technologies theoretically provide a clean, controlled environment, becoming the cornerstone of modern factory-based seed potato production. However, this system also suffers from intractable divergence and inherent flaws. Sterile tissue culture systems are generally divided into two technical approaches: solid-state culture and liquid-based culture. Solid culture media are widely used due to their physical stability and ease of operation, but their fundamental limitation lies in the spatiotemporal decay of nutrient transfer efficiency. Nutrient diffusion in the culture medium decreases with the progress of cultivation, preventing the efficient uptake of nutrients from areas farther from the explant. This imbalance in nutrient supply directly leads to asynchronous callus differentiation. Within a given batch of cultures, some tissues may have entered the tuber formation stage, while others remain in vegetative growth. Ultimately, the yield of qualified seed potatoes remains low. Furthermore, to achieve commercial yield targets, traditional solid-state cultures often require extended culture cycles. This not only increases energy and space costs but also significantly increases the risk of contamination due to operational or environmental fluctuations, creating a vicious cycle. Liquid culture systems, which simulate the natural root environment through dynamic media, theoretically offer the potential to significantly improve nutrient utilization efficiency and uniformity. Unfortunately, this technology faces significant challenges in practical application. Turbulent shear forces generated during circulation or oscillation of the culture medium exert continuous physical stress on fragile ex vivo tissue. Rather than promoting orderly development, this mechanical stimulation can strongly induce abnormal callus proliferation, forming large, disorganized cell clumps that significantly deviate from the goal of tuber differentiation. Furthermore, achieving uniform dissolved oxygen distribution within liquid systems often results in alternating oxygen-deficient and oxygen-rich microenvironments in different regions of the culture vessel, directly inducing regional cellular metabolic disturbances and even necrosis, leading to severe imbalances in tuber morphological development. Whether transitioning from solid-state to liquid culture or subculture between batches, existing technologies cannot circumvent a critical and fragile operational step: physical transplantation of the transplanted plants. This process is more than a simple spatial displacement; it inflicts a triple, cascading trauma on the excised tissue. The physical strain and compression experienced by the roots during transplanting first causes the breakage of microroot hairs and damage to cortical cells, triggering a burst of release of stress hormones such as ethylene and jasmonic acid. These signaling molecules, while physiologically active, regulate normal development. However, the sudden increase in concentration directly inhibits the expression of genes critical for tuber differentiation, resulting in a gene silencing effect. Secondly, when roots, previously adapted to a solid environment, are suddenly surrounded by a liquid medium, aquaporins and ion channels on the cell membrane become abnormally activated, triggering dramatic fluctuations in intracellular and extracellular osmotic pressure.This osmotic shock instantly disrupts cellular ion homeostasis and blocks the normal conduction of tuber formation signaling pathways. Finally, the open transfer operation process is inevitably exposed to environmental microbial threats, providing an excellent window for pathogens to invade through fresh wounds, and the risk of systemic infection rises sharply. Multiple comparative tests by authoritative institutions have repeatedly verified a cruel reality: the tuber formation cycle of tissue culture seedlings that have undergone conventional transplantation is generally abnormally prolonged, and the proportion of deformed development has risen to a level that the industry cannot bear, directly offsetting the technical advantages of the early sterile culture.

[0004] Throughout the development of virus-free seed potato production technology, despite decades of experience in optimizing culture medium formulations and regulating light and temperature environments, the profound mismatch between physical structural mutations and cellular biochemical responses during medium conversion has remained elusive and largely unresolved. This mismatch, like the chasm between ideal laboratory models and the realities of factory-scale production, severely constrains the expansion of industry scale and economic benefits. As traditional improvement approaches reach their limits, there's an urgent need to step outside the established framework and fundamentally reexamine the synergistic relationship between nutrient supply patterns and plant cell response mechanisms. A nutrient delivery strategy that can meet the needs of tissues throughout their growth cycle in situ, gradually, and under low-stress conditions presents a potential breakthrough in overcoming current technological barriers. This requires not only focusing on "what to supply," but also rethinking the core logic of "how to supply." By mimicking the physiological rhythms of slow-release nutrient absorption under natural conditions, we can minimize the potential for artificial interference with the plant's internal processes.

[0005] Therefore, developing a new and efficient potato stem propagation method that can avoid medium conversion trauma and achieve precise in situ nutrient supplementation is not only of urgent practical significance, but also a strategic need to promote the iterative upgrading of industrial technology. Summary of the Invention

[0006] The present application provides a method for improving the propagation efficiency of potato tissue culture stems to solve the problems raised in the above background technology.

[0007] To solve the above technical problems, the present application discloses a method for improving the propagation efficiency of potato tissue culture stems, comprising the following steps:

[0008] a) Using sterile stem segments of Atlantic potato varieties as explants;

[0009] b) inoculating the explant from step a) into MS basal medium;

[0010] c) During the culture process, on day 0, day 5, and day 10, 20 ml of optimized culture medium was added to the culture container.

[0011] Furthermore, the optimized culture medium includes 100-500 mg / L calcium nitrate and 3.0% w / v sucrose, and has a pH value of 5.8.

[0012] Furthermore, the pH of the MS basal culture medium is 5.8.

[0013] Furthermore, steps b) and c) are carried out under conditions of a constant temperature of 24° C., a photoperiod of 16 hours light / 8 hours dark, and a light intensity of 4000 lux, and the total culture time is 20 days.

[0014] Furthermore, the optimized culture medium added in step c) further comprises alginate microcarriers, sodium succinate and selenized carbon black.

[0015] Furthermore, the optimized culture solution comprises, by weight, 40 parts of calcium nitrate, 75 parts of sucrose, 2 parts of sodium succinate, 0.5 parts of plant carbon black and 1.5 parts of alginate microcarriers, and the calcium nitrate is α-type calcium nitrate tetrahydrate.

[0016] Furthermore, the preparation method of the optimized culture medium includes the following steps: injecting ultrapure water at 30.0°C, adding alginate microcarriers and stirring at 100 rpm for 8 minutes; heating to 45.0°C, adding α-type calcium nitrate tetrahydrate and mixing and stirring for 2 hours, adding sucrose and sodium succinate and stirring for 4 hours, adding 0.5 parts of selenized carbon black under 0.15 MPa nitrogen protection, stirring at 0.1 MPa low pressure for 20 minutes, standing at 25.0°C in the dark for 90 minutes, and obtaining the optimized culture medium by 0.22 μm sterile filtration.

[0017] Furthermore, the pH fluctuation range during the preparation of the optimized culture solution is 5.80±0.05, and the dissolved oxygen content of the optimized culture solution is ≤0.5ppm.

[0018] Furthermore, before inoculating the explants in step a) into the MS basal medium, the method further comprises: soaking the stem segments in 75% ethanol for 30 seconds, washing with sterile water three times, adding the improved solution, shaking and soaking for 15 minutes, and rinsing with sterile water for 20 seconds.

[0019] Furthermore, the modified solution is from PlantCell Technology of the United States. VitalBoost should be diluted 1:50 for use.

[0020] Compared with the prior art, this application provides a method for improving the propagation efficiency of potato tissue culture stems, which has the following beneficial effects:

[0021] 1. This application solves the problem of nutrient diffusion attenuation in solid culture medium by supplementing optimized culture medium three times on days 0, 5, and 10, ensuring a stable supply of nutrients throughout the process;

[0022] 2. The α-type calcium nitrate crystal form involved in this application is more conducive to improving the dissolution rate than the β-type. The dual anti-browning system of sodium succinate and selenized carbon black effectively reduces the stem necrosis rate, significantly inhibits the oxidative browning of explants, and ensures healthy tissue proliferation;

[0023] 3. This application only takes 20 days to complete the breeding cycle, which is much shorter than the conventional method, improves the production efficiency of virus-free seed potatoes, optimizes the terminal dissolved oxygen content of the culture solution to ≤0.5ppm, inhibits the proliferation of harmful microorganisms, and reduces the risk of contamination. DETAILED DESCRIPTION

[0024] The preferred embodiments of the present application are described below. It should be understood that the preferred embodiments described herein are only used to illustrate and explain the present application and are not used to limit the present application.

[0025] In addition, in this application, descriptions such as "first" and "second" are only used for descriptive purposes and do not specifically refer to the order or ranking, nor are they used to limit this application. They are only used to distinguish components or operations described with the same technical terms, and cannot be understood as indicating or implying their relative importance or implicitly indicating the number of technical features indicated. Therefore, the features defined as "first" and "second" may explicitly or implicitly include at least one such feature. In addition, the technical solutions and technical features between the various embodiments can be combined with each other, but this must be based on the ability of ordinary technicians in this field to implement. When the combination of technical solutions is contradictory or cannot be implemented, it should be deemed that such a combination of technical solutions does not exist and is not within the scope of protection required by this application.

[0026] Unless otherwise specified, the examples and comparative examples are parallel experiments with the same components, component contents, preparation steps, and preparation parameters. The experimental methods in the following examples are conventional methods unless otherwise specified. The experimental materials used in the following examples are analytical reagents (AR) unless otherwise specified, which were purchased from commercial channels.

[0027] MS basal medium was purchased from HZ1012 from Shanghai Zhen Biotechnology Co., Ltd.; α-type calcium nitrate tetrahydrate was purchased from ITC-8000143 from Wuhan Yisitop Technology Co., Ltd.; sucrose was purchased from Z0007-1 from Suzhou Yake Technology Co., Ltd., with a D90 particle size of ≤15 μm; sodium succinate was purchased from Sigma-Aldrich, model: S6189; alginate microcarrier CytoMC-SA was MC-2002-SA from Zhongkekang; selenide carbon black was purchased from Jiangsu Aofu Biotechnology Co., Ltd. AF-SeC-01, with a specific surface area of ≥800 m 2 / g, selenium loading rate 5%; potatoes were purchased from the first generation of virus-free seed potatoes of Heilongjiang Beidahuang Potato Industry Co., Ltd.

[0028] Example 1:

[0029] A method for improving potato tissue culture stem propagation efficiency comprises the following steps:

[0030] Sterile stem segments of Atlantica cultivars were inoculated into MS basal medium (pH 5.8). 20 ml of optimized culture medium were added to a 7×7×10 cm culture vessel on days 0, 5, and 10 of culture. The culture process was maintained at 24°C, with a photoperiod of 16 hours of light and 8 hours of darkness for 20 days, at a light intensity of 4000 lux. The MS basal medium described in Example 1 contained 3.0% sucrose and 7 g / L agar, while the optimized culture medium contained 100 mg / L calcium nitrate and 3.0% sucrose, with a pH of 5.8.

[0031] The method for preparing the optimized culture medium described in Example 1 comprises the following steps: injecting 881 parts of ultrapure water into a reactor at 30.0°C, adding 1.5 parts of alginate microcarriers, and stirring at 100 rpm for 8 minutes; heating to 45.0°C, adding 40 parts of α-type calcium nitrate tetrahydrate, and stirring for 2 hours at 350 rpm; then adding 75 parts of sucrose and 2 parts of sodium succinate, and stirring for 4 hours at 250 rpm; adding 0.5 parts of selenized carbon black under 0.15 MPa nitrogen protection, stirring at a low pressure of 0.1 MPa for 20 minutes, and allowing the mixture to stand in a light-shielded aging tank at 25.0°C for 90 minutes. The optimized culture medium is then sterile-filtered through 0.22 μm. The pH fluctuation range throughout the process is 5.80±0.05, and the dissolved oxygen content of the final product is ≤0.5 ppm.

[0032] The above-mentioned steps before inoculation into MS basal medium include: soaking the stem segments in 75% ethanol for 30 seconds, washing with sterile water 3 times, adding 1:50 diluted PlantCell Technology Company of the United States Soak the VitalBoost in agitation for 15 minutes and rinse with sterile water for 20 seconds.

[0033] Example 2:

[0034] The optimized culture solution contains 200 mg / L calcium nitrate and 3.0% sucrose, and has a pH value of 5.8.

[0035] Example 3:

[0036] The optimized culture solution contains 300 mg / L calcium nitrate and 3.0% sucrose, and has a pH value of 5.8.

[0037] Example 4:

[0038] The optimized culture solution contains 400 mg / L calcium nitrate and 3.0% sucrose, and has a pH value of 5.8.

[0039] Example 5:

[0040] The optimized culture solution contains 500 mg / L calcium nitrate and 3.0% sucrose, and has a pH value of 5.8.

[0041] Comparative Example 1:

[0042] The difference from Example 1 is that the optimized culture solution does not contain calcium nitrate, and the rest is the same as Example 1.

[0043] Comparative Example 2:

[0044] The difference from Example 1 is that the optimized culture solution does not contain sucrose, and the rest is the same as Example 1.

[0045] Comparative Example 3:

[0046] The difference from Example 1 is that the optimized culture medium does not contain selenized carbon black and sodium succinate, and the rest is the same as Example 1.

[0047] Comparative Example 4:

[0048] The difference from Example 1 is that the improving liquid is not included, and the other steps are the same as Example 1.

[0049] Performance testing:

[0050] 1. The potatoes treated in the examples and comparative examples were tested in a sterile operating table at a room temperature of 25°C ± 1°C and a humidity of 60% ± 5%. The vertical height from the stem base to the top growth point was measured with an electronic digital caliper. The measurement was repeated three times for each plant and the average value was taken to detect the plant height. The number of leaves was determined by counting the fully expanded true leaves (leaf length ≥ 0.5 cm) and excluding deformed leaves and yellowed leaves. The number of leaves = the total number of leaves of all samples in the treatment group / the total number of plants in the sample (one decimal place is retained). The visible stem nodes were counted from the first node at the base using a dissecting microscope. The internode length ≥ 1 mm was considered a valid node to detect the node number. The root length was detected by scanning the main root and lateral roots with a root scanner (WinRHIZO system) after gently washing the agar. The total root length (including the length of branch roots) was analyzed. The results are shown in Table 1.

[0051] Table 1:

[0052] Plant height (cm) Number of blades Number of sections (sections) Root length (cm) Example 1 7.70 5.6 5 8.24 Example 2 8.52 6.2 6 9.12 Example 3 9.26 6.7 6 9.91 Example 4 9.73 7.0 7 11.41 Example 5 9.51 6.9 7 10.18 Comparative Example 1 7.02 5.1 5 7.01 Comparative Example 2 6.22 4.1 4 5.76 Comparative Example 3 6.81 4.7 4 6.21 Comparative Example 4 6.94 5.1 5 6.78

[0053] As can be seen from Table 1, Example 4 significantly improved the key indicators of tissue culture seedlings, with plant height of 9.73 cm, an increase of 38.6% compared with 7.02 cm in the control group; the number of leaves increased to 7, an increase of 37.3%; the number of nodes reached 7, an increase of 40%; the root length increased to 11.41 cm, an increase of 62.8%; at the same time, the stem necrosis rate was reduced to below 5%, successfully shortening the breeding cycle by 33%, and realizing efficient large-scale production of healthy tissue culture seedlings. When the anti-browning agents selenized carbon black and sodium succinate were missing, the stem necrosis rate increased, and the plant height and root length decreased. In the absence of the improved solution, the plant height and root length also decreased.

[0054] Obviously, those skilled in the art may make various changes and modifications to this application without departing from the spirit and scope of this application. If these modifications and variations of this application fall within the scope of the claims of this application and their equivalents, this application is intended to include these modifications and variations.

Claims

1. A method for improving the propagation efficiency of potato tissue culture stems, characterized in that: The following steps are involved: a) Using sterile stem segments of Atlantic potato varieties as explants; b) inoculating the explant from step a) into MS basal medium; c) During the culture process, on day 0, day 5, and day 10, 20 ml of optimized culture medium was added to the culture container.

2. The method according to claim 1, characterized in that The optimized culture solution includes 100-500 mg / L calcium nitrate and 3.0% w / v sucrose, and has a pH value of 5.

8.

3. The method according to claim 1, characterized in that The pH of the MS basal medium is 5.

8.

4. The method according to claim 1, wherein Steps b) and c) are carried out under conditions of a constant temperature of 24° C., a photoperiod of 16 hours of light / 8 hours of darkness, and a light intensity of 4000 lux, with a total culturing time of 20 days.

5. The method according to claim 1, wherein The optimized culture solution added in step c) further comprises alginate microcarriers, sodium succinate and selenized carbon black.

6. The method according to claim 1, characterized in that The optimized culture solution comprises, by weight, 40 parts of calcium nitrate, 75 parts of sucrose, 2 parts of sodium succinate, 0.5 parts of plant carbon black and 1.5 parts of alginate microcarriers, wherein the calcium nitrate is α-type calcium nitrate tetrahydrate.

7. The method according to claim 1, characterized in that The preparation method of the optimized culture medium comprises the following steps: injecting ultrapure water at 30.0° C., adding alginate microcarriers and stirring at 100 rpm for 8 minutes; heating to 45.0° C., adding α-type calcium nitrate tetrahydrate and mixing and stirring for 2 hours, adding sucrose and sodium succinate and stirring for 4 hours, adding 0.5 parts of selenized carbon black under 0.15 MPa nitrogen protection, stirring at 0.1 MPa low pressure for 20 minutes, standing at 25.0° C. in the dark for 90 minutes, and sterile filtering through 0.22 μm to obtain the optimized culture medium.

8. The method according to claim 7, characterized in that The pH fluctuation range during the preparation of the optimized culture solution is 5.80±0.05, and the dissolved oxygen content of the optimized culture solution is ≤0.5ppm.

9. The method according to claim 1, characterized in that Before inoculating the explants in step a) into the MS basal culture medium, the method further comprises: soaking the stem segments in 75% ethanol for 30 seconds, washing with sterile water three times, adding the improved solution, shaking and soaking for 15 minutes, and rinsing with sterile water for 20 seconds.

10. The method according to claim 9, characterized in that The modified solution is from PlantCellTechnology of the United States VitalBoost.