A method for improving the efficiency of potato tissue culture stem propagation

Abstract

The application provides a method for improving the propagation efficiency of potato tissue culture stems, which comprises the following steps: taking an aseptic stem segment of Atlantic variety as an explant, inoculating into a pH 5.8 MS basic culture medium containing 3.0% sucrose and 7 g / L agar, supplementing the culture container with an optimized culture solution containing 100 mg / L calcium nitrate and 3.0% sucrose for three times at the 0th, 5th and 10th days of culture, culturing for 20 days under the conditions of 24 DEG C, 16 h light / 8 h darkness and 4000 lux, using alginate microcarriers to load active ingredients, adding selenium carbon black and sodium succinate under the protection of nitrogen through staged temperature control stirring, preparing through 25 DEG C light-shielded maturation and sterile filtration, and the terminal dissolved oxygen content is less than or equal to 0.5 ppm. The method significantly shortens the culture period of potato stems, improves the propagation efficiency, and is suitable for large-scale production of virus-free seed potatoes.

Description

TECHNICAL FIELD

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

[0002] As a global important food and economic crop, the industrial value of potato has long exceeded the scope of simple food, and has a profound impact on the sustainable development pattern of food processing, bioenergy and modern agriculture. However, the core pain point of potato industry, the stable and efficient supply of healthy seed potatoes, has long been subject to the inherent technical bottlenecks in the seed potato propagation link. Among them, the industrialized production of virus-free seed potatoes is particularly crucial, as it not only concerns the yield potential, but also directly determines the quality grade and disease resistance of commodity potatoes. The current mainstream production mode mainly relies on two systems of greenhouse field breeding and tissue culture technology, but both are deeply trapped in the sharp contradiction between efficiency and cost.

[0003] Greenhouse production system can achieve a certain scale of seed potato output, but it is difficult to avoid repeated infection of pests and diseases. Soil or vector organisms carrying pathogens can easily accumulate in the generation alternation, leading to the rapid decline of detoxification advantage. More disturbingly, its production capacity is highly dependent on natural climate rhythm, and the light cycle and temperature change cannot be precisely controlled by human beings, resulting in a long production cycle and significant quality fluctuations between batches. In contrast, the technical route based on aseptic tissue culture can theoretically provide clean and controllable environmental protection, and has become the cornerstone of modern industrial production of seed potato. However, there are still route differentiation and inherent defects in the system. The aseptic tissue culture system is usually divided into solid culture and liquid culture. Solid medium is widely used due to its physical stability and operational convenience, but its essential limitation is the spatial and temporal decline of nutrient transfer efficiency. The nutrient diffusion in the medium shows a gradient decline trend with the progress of culture, and the nutrients in the area far from the explant cannot be efficiently taken up. This non-uniformity of nutrient supply directly leads to asynchronous differentiation of callus, and part of the culture may have entered the tuber formation stage, while the other part is still in the vegetative growth state, resulting in a low proportion of qualified seed potato. More seriously, traditional solid culture is often forced to extend the culture period to achieve commercial production goals, which not only increases energy consumption and space cost, but also significantly increases the risk of contamination caused by operation or environmental fluctuations, forming a vicious cycle. Liquid culture system simulates the natural root environment through dynamic medium, which can theoretically greatly improve the efficiency and uniformity of nutrient utilization. Unfortunately, this technology has encountered insurmountable fluid mechanics obstacles in practical application. The turbulent shear force generated during the circulation or oscillation of the culture solution causes persistent physical stress on the fragile in vitro tissue. This mechanical stimulation not only cannot promote orderly development, but also strongly induces abnormal proliferation of callus, forming a large number of structurally disordered cell clusters, which deviate seriously from the tuber differentiation goal. At the same time, the distribution of dissolved oxygen in the liquid system is difficult to homogenize, and different areas of the culture container often form alternating anoxic and oxygen-rich microenvironments, directly inducing regional metabolic disorders and even necrosis, causing serious imbalance in tuber morphological development. Whether it is the transition from solid culture to liquid culture, or the transfer of different batches, the existing technology cannot avoid a key and fragile operation step—physical transfer of explants. This process is not simply a spatial displacement, but a triple-linked traumatic attack on in vitro tissue. The physical pulling and squeezing of roots during the transfer operation first causes the breakage of root hairs and the damage of cortical cells, triggering the explosive release of stress hormones such as ethylene and jasmonic acid. At physiological concentrations, these signal molecules can regulate normal development, but at high concentrations, they directly inhibit the expression of key genes involved in tuber differentiation, forming a gene silencing effect. Secondly, when the roots adapted to the solid environment are suddenly surrounded by liquid medium, the water channels and ion channels on the cell membrane are abnormally activated, causing a dramatic shock in the osmotic pressure between cells and the outside.This osmotic shock disturbs the ion homeostasis of cells instantly, and blocks the normal conduction of the signal pathway of tuber formation. Finally, the open transfer process is inevitably exposed to environmental microbial threats, providing an excellent window for pathogenic bacteria to invade through the fresh wound, and the risk of systemic infection rises sharply. Multiple comparative tests by authoritative agencies have repeatedly verified a harsh reality: the tuber formation period of tissue culture seedlings that have undergone regular transplanting is generally abnormally prolonged, and the proportion of abnormal development has climbed to an unaffordable level for the industry, directly offsetting the technical advantages of early aseptic culture.

[0004] Throughout the development process of potato virus-free seed potato production technology, although decades of experience have been accumulated in medium formula optimization and light and temperature environment regulation research, the physical structure mutation exposed during medium conversion and the deep mismatch of cellular biochemical response have always failed to be systematically recognized and fundamentally solved. This mismatch is like a gap between the ideal model in the laboratory and the reality of factory production, which seriously restricts the improvement of industrial scale and economic benefits. When the traditional improvement path reaches the ceiling, it is urgent to break out of the existing framework and fundamentally re-examine the synergistic relationship between nutrient supply mode and plant cell response mechanism. A nutrient delivery strategy that can meet the needs of the whole growth period of the tissue in situ, gradually, and under low stress conditions, becomes a potential breakthrough to break through the current technical predicament. This requires us not only to focus on "what to supply", but also to innovate the core logic of "how to supply", to simulate the physiological rhythm of nutrient release and absorption in natural state, and to avoid the rough disturbance of artificial intervention to the internal program of plants to the greatest extent.

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

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

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

[0008] a) using aseptic stem segments of Atlantic variety potatoes as explants;

[0009] b) inoculating the explants of step a) into MS basic medium;

[0010] c) during the culture process, adding optimized culture solution to the culture container on day 0, day 5 and day 10, respectively, with an addition amount of 20 ml each time.

[0011] Further, the optimization culture solution comprises 100-500 mg / L calcium nitrate and 3.0% w / v sucrose, and has a pH value of 5.8.

[0012] Further, the MS basic culture medium has a pH value of 5.8.

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

[0014] Further, the optimization culture solution added in step c) further comprises alginate microcarriers, sodium succinate, and carbon black selenium.

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

[0016] Further, the preparation method of the optimization culture solution comprises the following steps: injecting ultrapure water at 30.0°C, adding alginate microcarriers and stirring at 100 rpm for 8 minutes; increasing the temperature to 45.0°C, adding α-type calcium nitrate tetrahydrate, mixing and stirring for 2 hours, adding sucrose and sodium succinate, stirring for 4 hours, adding 0.5 parts of carbon black selenium under the protection of 0.15 MPa nitrogen, stirring at 0.1 MPa low pressure for 20 minutes, placing in the dark at 25.0°C for 90 minutes, and obtaining the optimization culture solution through sterile filtration through a 0.22 μm filter.

[0017] Further, the pH value fluctuation range in the preparation process of the optimization culture solution is 5.80±0.05, and the dissolved oxygen content of the optimization culture solution is ≤0.5 ppm.

[0018] Further, before inoculating the explants of step a) into the MS basic culture medium, the method further comprises the following steps: soaking the stem section in 75% ethanol for 30 seconds, washing with sterile water for 3 times, adding a modified liquid for oscillation and soaking for 15 minutes, and rinsing with sterile water for 20 seconds.

[0019] Further, the modified liquid is VitalBoost of Plant Cell Technology Company, USA, which is used after being diluted by 1:50. VitalBoost, which is used after being diluted by 1:50.

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

[0021] 1. The application solves the problem of nutrient diffusion attenuation of solid culture medium by supplementing the optimization culture solution three times on the 0th, 5th, and 10th days, and ensures stable nutrient supply throughout the process.

[0022] 2, The alpha type calcium nitrate crystal form is more conducive to improving the dissolution rate than the beta type, the sodium succinate and selenium carbon black double anti-browning system effectively reduces the stem necrosis rate and significantly inhibits the oxidation browning of explants, thereby ensuring the healthy proliferation of tissues;

[0023] 3, The propagation cycle of the application can be completed in only 20 days, which is greatly shortened compared with the conventional method, thereby improving the production efficiency of virus-free seed potatoes, and the terminal dissolved oxygen content of the culture solution is less than or equal to 0.5 ppm, thereby inhibiting the proliferation of harmful microorganisms and reducing the risk of bacterial contamination. DETAILED DESCRIPTION

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

[0025] In addition, the descriptions such as "first", "second" and the like in the present application are only for the purpose of description, and do not mean to indicate the order or sequence, nor to limit the application, which is only to distinguish the components or operations described by the same technical terms, and cannot be understood as indicating or implying the relative importance or implicitly indicating the number of the indicated technical features. Therefore, the features limited by "first" and "second" can be explicitly or implicitly included at least one of the features. In addition, the technical solutions and technical features of various embodiments can be combined with each other, but it must be based on the realization of ordinary skilled in the art, when the combination of technical solutions appears contradictory or unachievable, it should be considered that the combination of technical solutions does not exist, nor within the scope of protection claimed by the present application.

[0026] Unless otherwise specified, the examples and comparative examples are parallel tests 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 test materials used in the following examples are analytical reagents (A.R.) unless otherwise specified, which are purchased from commercial channels.

[0027] The MS basic medium is purchased from HZ1012 of Shanghai Zhen Biological Technology Co., Ltd.; the alpha type calcium nitrate tetrahydrate is purchased from ITC-8000143 of Wuhan Yisitupu Technology Co., Ltd.; the sucrose is purchased from Z0007-1 of Suzhou Yakexin Technology Co., Ltd., with a D90 particle size of ≤15 μm; the sodium succinate is purchased from Sigma-Aldrich, model number: S6189; the alginate microcarrier CytoMC-SA is MC-2002-SA of Zhongke Kang; the selenium carbon black is AF-SeC-01 of Jiangsu Aofu Biological Technology, with a specific surface area of ≥800 m 2 / g and a selenium loading rate of 5%; the potatoes are 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 the efficiency of potato tissue culture stem propagation, comprising the following steps:

[0030] Using aseptic stem segments of Atlantic variety as material, inoculating in MS basic medium with pH 5.8, and adding 20 ml of optimized culture solution each time at 0, 5, 10 days of culture in 7×7×10 cm culture containers, the whole culture process is carried out at 24℃ constant temperature, 16 hours light, 8 hours darkness for 20 days, and the light intensity is 4000 lux. The MS basic medium described in the above embodiment 1 contains 3.0% sucrose and 7 g / L agar, and the optimized culture solution contains 100 mg / L calcium nitrate and 3.0% sucrose, with pH value of 5.8.

[0031] The preparation method of the optimized culture solution described in the above embodiment 1 comprises the following steps: injecting 881 parts of ultrapure water into a reaction kettle at 30.0℃, adding 1.5 parts of alginate microcarrier and stirring at 100 rpm for 8 minutes; after heating to 45.0℃, adding 40 parts of α-type calcium nitrate tetrahydrate and mixing and stirring for 2 hours at a speed of 350 rpm, adding 75 parts of sucrose and 2 parts of sodium succinate and stirring for 4 hours at a speed of 250 rpm; under the protection of 0.15 MPa nitrogen, adding 0.5 parts of carbon black selenide, stirring at 0.1 MPa low pressure for 20 minutes, and standing in a light-proof aging tank at 25.0℃ for 90 minutes, and then filtering through a 0.22 μm sterile filter to obtain the optimized culture solution. The pH fluctuation range is 5.80±0.05 throughout the process, and the dissolved oxygen content of the final product is ≤0.5 ppm.

[0032] The above inoculation in the MS basic medium further comprises the following steps: soaking the stem segments in 75% ethanol for 30 seconds, washing with sterile water for 3 times, adding 1:50 diluted VitalBoost of American Plant Cell Technology Company, and oscillating soaking for 15 minutes, and rinsing with sterile water for 20 seconds. VitalBoost oscillating soaking for 15 minutes, and rinsing with sterile water for 20 seconds.

[0033] Example 2:

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

[0035] Example 3:

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

[0037] Example 4:

[0038] The optimized culture solution contains 400 mg / L calcium nitrate and 3.0% sucrose, with 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 solution does not contain selenium 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 modified solution is not included, and the rest is the same as Example 1.

[0049] Performance test:

[0050] I. Test the potatoes treated in the examples and comparative examples in a sterile operation table, the room temperature is 25℃±1℃, the humidity is 60%±5%, the vertical height from the stem base to the top growing point is measured with an electronic digital caliper, the average value of three repeated measurements per plant is taken to detect the plant height, the fully expanded true leaves (leaf length≥0.5cm) are counted and the abnormal leaves and yellow leaves are excluded to test the leaf number, the leaf number = the total number of leaves of all samples in the treatment group / the total number of plants (retaining one decimal place), the visible stem nodes are counted from the first node at the base with a dissecting microscope, and the inter-node length≥1mm is regarded as an effective node to detect the node number, and the main root and lateral root are scanned after gentle washing with agar with a root scanning instrument (WinRHIZO system), and the total root length (including branch root length) is analyzed to detect the root length, and the results are shown in Table 1.

[0051] Table 1:

[0052] Plant height (cm) Leaf number (piece) Node number (node) 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 shown in Table 1, the key indicators of the tissue culture seedlings of Example 4 were significantly improved, the plant height reached 9.73 cm, increased by 38.6% compared with 7.02 cm of the control group; the leaf number increased to 7, increased by 37.3%; the node number reached 7, increased by 40%; the root length reached 11.41 cm, increased by 62.8%; at the same time, the stem necrosis rate was reduced to less than 5%, the propagation cycle was shortened by 33%, the healthy tissue culture seedlings were efficiently and scaled up, when the anti-browning agent selenium black and sodium succinate were missing, the stem necrosis rate increased, the plant height and root length decreased, and when there was no improvement liquid, the plant height and root length also decreased.

[0054] Obviously, those skilled in the art can make various modifications and variations to the present application without departing from the spirit and scope of the present application. If these modifications and variations of the present application fall within the scope of the claims of the present application and their equivalents, the present application also intends to include these modifications and variations.

Claims

1. A method for improving the efficiency of potato tissue culture shoot propagation, characterized by, The method comprises the following steps: a) taking aseptic stem segments of Atlantic variety potato as explants; b) inoculating the explants of step a) in MS basic medium; c) during the culturing process, adding 20 ml of optimized culture solution to the culturing container at the 0th day, 5th day and 10th day, respectively; the optimized culture solution is composed of the following components in parts 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 microcarrier, and the calcium nitrate is alpha type calcium nitrate tetrahydrate.

2. The method of claim 1, wherein, The pH of the MS basic medium is 5.

8.

3. The method of claim 1, wherein, Steps b) and c) are carried out under the conditions of constant temperature of 24 DEG C, light cycle of 16 hours light / 8 hours dark, light intensity of 4000 lux, and total culturing time of 20 days.

4. The method of claim 1, wherein, The preparation method of the optimized culture solution comprises the following steps: injecting ultrapure water at 30.0 DEG C, adding alginate microcarriers and stirring at 100 rpm for 8 minutes; increasing the temperature to 45.0 DEG C, adding alpha type calcium nitrate tetrahydrate and mixing for 2 hours, adding sucrose and sodium succinate and stirring for 4 hours, adding 0.5 parts of selenium carbon black under the protection of 0.15 MPa nitrogen, stirring at 0.1 MPa low pressure for 20 minutes, placing in the dark at 25.0 DEG C for 90 minutes, and sterilizing through 0.22 mu m sterile filtration to obtain the optimized culture solution.

5. The method of claim 4, wherein, 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 less than or equal to 0.5 ppm.

6. The method of claim 1, wherein, Before inoculating the explants of step a) in the MS basic medium, the method further comprises the following steps: soaking the stem segments in 75% ethanol for 30 seconds, washing with sterile water for 3 times, adding modified solution and oscillating for 15 minutes, and rinsing with sterile water for 20 seconds. The modified solution is VitalBoost of Plant Cell Technology Company, USA.