A method for producing indoor seed tubers using solid and liquid culture media

Abstract

The application provides a method for producing indoor seed potatoes by using solid culture medium and liquid culture medium, and the method comprises the following steps: in the initial stage, potato stem segments are inoculated by using a solid culture medium containing 3.0% sucrose and 10 g / L agar, and the inoculated potato stem segments are cultured at 24 DEG C under 4000 lux illumination (16 hours / day) for 25 days; 25 ml of liquid culture solution is added; in the later stage, the potato stem segments are transferred into a tuber induction environment, the humidity is maintained through a microporous air permeable film, and double-waveband light signal combination is applied to synergistically promote tuber formation and enlargement; the tuber induction rate and the weight of single potato are significantly improved through accurate regulation of the photoperiod and dynamic switching of the culture medium; the whole process is completed in a closed container, the re-invasion of viruses is effectively prevented, the malformation rate and the moldy rate of the produced potatoes are low, and the method is suitable for indoor production of virus-free seed potatoes.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of potato, in particular to a method for producing indoor seed potatoes by using solid medium and liquid medium. BACKGROUND

[0002] Potato, commonly known as potato, egg, and potato, is a Solanaceae L. annual herbaceous plant, originally from the Andes Plateau in South America, and the artificial cultivation history can be traced back to the southern Peru from 8000 BC to 5000 BC. In the mid-16th century, Spanish colonists introduced it to Europe, initially as an ornamental plant, and later because of the promotion of its edible value by French agronomist Anne Obamanchi, it gradually became an important global food crop. It was introduced to China in the 17th century and has been planted for more than 300 years. Potato is the fourth largest food crop in the world, second only to rice, wheat and corn. China is the largest producer, with planting area and yield accounting for more than one-fourth of the world, mainly distributed in the north one-cropping area, central China two-cropping area, southwest mixed cropping area and southern winter cropping area, which can be processed into potato chips, starch, biofuel, etc., and has wide industrial applications. Since the commercialization of the virus-free rapid propagation technology of potato seed potatoes in the 1970s, there have been two major contradictions: the antagonistic balance between sterile environment control and large-scale production cost. In addition, the industrialized production of virus-free seed potatoes has long relied on two basic systems of greenhouse field production and natural photoperiod induction for tuber formation, which has a risk of cross-infection of diseases and pests and is subject to seasonal constraints. The sterile tissue culture system is divided into solid and liquid culture paths. Although the solid medium can guarantee the sterile environment, the nutrient diffusion efficiency significantly decreases with the extension of the culture time, resulting in poor synchronization of tuber differentiation and low output rate of qualified seed potatoes. The liquid culture system improves the efficiency of nutrient utilization, but the turbulent shear force easily causes abnormal proliferation of callus, and uneven distribution of dissolved oxygen causes regional necrosis of cells, resulting in imbalance of tuber morphological development. In the key link of conversion from solid to liquid culture, the existing technology needs to physically transfer the plant. This process causes three linked damages: root system transfer operation causes microstructure damage, triggers explosive release of stress hormones, directly inhibits mechanical trauma of tuber differentiation gene expression; when the root system suddenly encounters a liquid environment, the cell membrane channel protein is instantaneously activated, causing a dramatic change in ion concentration, blocking the osmotic shock of the tuber formation signal pathway; open transfer operation greatly increases the risk of contamination, and pathogenic bacteria invade through the wound, causing a microbial infection window of systemic bacterial infection. This phenomenon shows that although medium formula optimization and light and temperature control research have been ongoing for decades, the problem of mismatch between physical structure mutation and biochemical response during medium conversion has not been systematically recognized. International authoritative agencies have confirmed that conventional transfer operation causes abnormal prolongation of the tuber formation period, and the rate of deformity rises to an unacceptable level in the industry.

[0003] Therefore, it is of great significance to develop a new method for producing indoor seed potatoes by using solid medium and liquid medium. SUMMARY

[0004] The present application provides a method for producing indoor seed potatoes by using solid medium and liquid medium to solve the problems in the background art.

[0005] To solve the above technical problems, the present application discloses a method for producing indoor seed potatoes by using solid medium and liquid medium, comprising the following steps:

[0006] S1. Initial culture stage: inoculate potato stem segments into solid medium and culture for 25 days;

[0007] S2. Liquid medium addition: add 25 ml of liquid culture solution;

[0008] S3. Late culture stage: culture for 80 days under the conditions of 4 hours of light and 20 hours of darkness, and maintain the humidity at 85-90%.

[0009] Further, inoculate potato stem segments into solid medium, and culture for 25 days under the conditions of temperature 24±1℃, light intensity 4000 lux, 16 hours of light and 8 hours of darkness.

[0010] Further, the solid medium is 1 / 2MS basic medium, with the addition of 3.0% sucrose and 10 g / L agar, and the pH value is 5.7.

[0011] Further, the liquid culture solution is 1 / 4MS basic medium, with the addition of 9.0% sucrose, and the pH value is 6.2.

[0012] Further, place the solid medium in a culture container with the size of 7 cm×7 cm×10 cm, and inoculate 6 stem segments into each culture container.

[0013] Further, the late culture stage further comprises light regulation, which comprises 4 hours of 400-500 nm blue light as the main light source every day.

[0014] Further, the late culture stage further comprises light regulation, which further comprises 660 nm red light pulse as the auxiliary light source, 1-3 times a day, and 15-25 minutes each time.

[0015] Further, the potato stem segments are single-node stem segments of virus-free test tube seedlings, with the length of 0.5-1.0 cm.

[0016] Further, the late culture stage further comprises injecting 25 ml of optimized culture solution into the bottom of the culture container on the first day of the late culture stage, with the injection position being the liquid injection hole reserved on the side wall of the container, the injection needle being inserted at an angle of 45°, and the control flow rate being 2 ml / min. After injection, stand for 10 minutes, and promote the uniform diffusion of the culture solution to the solid medium matrix by oscillation.

[0017] Further, the optimization culture solution comprises the following components: 1 / 4MS base mother liquor, 90 g / L sucrose, 500 mg / L hydrolyzed casein, 2 g / L trehalose, 0.01% polylactic acid-hydroxy acid copolymer coated ATP-bushen acid sodium nanomicelles, and 0.1 mg / L paclobutrazol; the pH value of the optimization culture solution is 6.0±0.05; the 1 / 4MS base mother liquor is prepared by doubling the KNO3 content to 3.8 g / L, dissolving and stirring at 40±2°C for 30 minutes, and storing at 4°C in the dark after filtration; the ATP-bushen acid sodium nanomicelles are prepared by the following steps: dissolving 5 mg of ATP-Na2 and 0.05 mg of bushen acid in 10 mL of ultrapure water, ultrasonically treating at a power of 200 W and a frequency of 40 kHz for 15 minutes in an ice bath at 4°C, and stirring at 150 rpm for 2 hours after adding 0.01% polylactic acid-hydroxy acid copolymer.

[0018] Compared with the prior art, the application provides a method for producing indoor seed potatoes by using solid culture medium and liquid culture medium, which has the following beneficial effects:

[0019] 1. The two-stage culture medium design of the application synergistically promotes growth and tuber differentiation, the initial stage uses solid culture medium to provide stable support and nutrient basis for stem segments and ensure early root development, and the later stage adds liquid culture solution to accelerate the translocation of photosynthetic products to tubers through osmotic pressure;

[0020] 2. The light period and spectrum regulation of the application accurately induces tuber formation, the later stage uses short-day light combined with double-waveband light signals, the main light source blue light regulates photomorphogenesis and promotes the expression of tuber initiation genes, and the pulsed red light is irradiated for a short time 1-3 times a day to activate the phytochrome PhyB and enhance the activity of starch synthase, and the light shield in the dark period blocks external light sources to ensure 20 hours of continuous dark period and maximize the accumulation of tuber formation hormones;

[0021] 3. The environmental parameter optimization of the application reduces physiological stress, the later stage is close to the optimum temperature for tuber swelling at 20°C, inhibits stem and leaf growth and promotes the allocation of assimilates to tubers, the microporous breathable film balances oxygen exchange and transpiration inhibition, prevents high humidity from causing callus, and prevents low humidity from causing dehydration; in addition, the space utilization rate and standardized production advantage, the culture container specifications are uniform, 6 stem segments are inoculated in each container, and the dense arrangement can significantly improve the yield of seed potatoes per unit area; the whole process is controlled in an environment to get rid of seasonal restrictions and realize year-round production, which shortens the traditional greenhouse seedling raising cycle by more than 30%;

[0022] 4、The application relates to an optimized culture solution nanotechnique for improving biological activity, through polylactic acid-glycolic acid copolymer encapsulation, enhanced stability of small molecule active substances, prolonged release time; ice bath ultrasonic breaking of molecular aggregation, improved cell membrane permeability, promoted efficient absorption of energy substances and antioxidant components, accelerated tuber cell division; precise liquid injection process ensures root safety and nutrition homogeneity, 45° side wall liquid injection avoids direct impact on the surface of the culture medium, prevents stem displacement or damage; low-speed injection combined with oscillation diffusion ensures uniform penetration of the liquid into the solid culture medium matrix, eliminates salt stress caused by local high concentration; after sterilization, the active multi-effect paclobutrazol dissolved in ethanol inhibits gibberellin synthesis, further prevents overgrowth and promotes tuber enlargement. DETAILED DESCRIPTION

[0023] 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.

[0024] In addition, the description of "first", "second" and the like in the present application is only for the purpose of description, and does not mean to specially 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 of the indicated technical features or implicitly indicating the number of the indicated technical features. Therefore, the features limited by "first", "second" can explicitly or implicitly include 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.

[0025] 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.

[0026] 1 / 4MS was purchased from Qingdao Haibo Biology HB8469-8; sucrose was purchased from Xilong Scientific, purity ≥ 99.5%; hydrolyzed casein was purchased from Shanghai Jituo Biochemical Technology CAS 65072-00-6; ATP-Na2 was purchased from Shanghai Solabio Biological Technology A8270-1; Shikonin was purchased from Shandong Xiya Chemical B20610; Paclobutrazol was purchased from Shanghai Zhenzhun Biological Technology Paclobutrazol Mixture (ZZS-46046-250MG); Polylactic acid-glycolic acid copolymer was purchased from Hangzhou Xinjiao Biological Technology PLGA-PEGMW:5000 (50:50 ratio); Trehalose was purchased from Shanghai Jituo Biochemical Technology CAS 99-20-7.

[0027] Example 1:

[0028] A method for producing indoor seed potatoes using solid and liquid culture media includes the following steps:

[0029] 1. Initial culture stage (25 days): A solid culture medium with pH 5.7 is prepared using 1 / 2MS + 3.0% sucrose + 10 g / L agar, and the culture medium is placed in a culture container with a size of 7 cm x 7 cm x 10 cm. Six potato stem segments are aseptically inoculated into each such container. The entire initial culture process is carried out in a strictly controlled environment, with the culture temperature maintained at 24°C, the light cycle set to 16 hours of light per day combined with 8 hours of darkness, and the light intensity maintained at 4,000 lux to promote the healthy growth and development of the stem segments.

[0030] Liquid culture medium addition: A liquid culture medium with pH 6.2 is prepared using 1 / 4MS + 9.0% sucrose, and 25 ml of the liquid culture medium is added to the culture container after the initial culture stage.

[0031] Late culture stage (80 days): In order to fully induce and optimize tuber formation and enlargement, the culture temperature is strictly maintained at 20°C, and the light cycle is changed to 4 hours of 2000 lux white light illumination per day and 20 hours of darkness. The light shield completely blocks external light sources during the dark period, ensuring that the short-day effect is maximized to induce tuber formation. The relative humidity in the container is maintained at 85-90% through a microporous breathable film, which not only reduces transpiration stress but also ensures oxygen exchange required for tuber respiration during the dark period.

[0032] In order to utilize the regulatory mechanism of light environment in the above examples, the late culture stage also includes embedding a dual-band light signal combination in the short-day cycle, which includes a main light source that lasts for 4 hours per day and 400-500 nm blue light, and 660 nm red light as an auxiliary pulse that is intermittently irradiated 1-3 times per day, each time for 15-25 minutes.

[0033] Example 2:

[0034] Late culture stage (80 days): In order to fully induce and optimize the formation and enlargement of tubers, 25 ml of optimized culture solution was injected into the bottom of the culture container on the first day of the late culture stage, the injection position was the reserved injection hole on the side wall of the container, the injection needle was inserted at an angle of 45°, and the control flow rate was 2 ml / min. After injection, stand for 10 minutes, oscillation promotes the uniform diffusion of the culture solution to the solid culture medium matrix, strictly maintain the culture temperature at 20℃, the light cycle is changed to 4 hours of 2000 lux white light illumination per day and 20 hours of darkness. The light shield completely blocks the external light source to ensure that the short-day effect maximizes the induction of tuber formation, the relative humidity in the container is maintained at 85-90% through the microporous breathable film, which not only reduces the transpiration stress, but also ensures the oxygen exchange required for tuber respiration in the dark period, and other contents are consistent with Example 1.

[0035] The method of the above-mentioned optimized culture solution used in Example 2 includes:

[0036] S1, mother liquor pre-dispensing: In a sterile laboratory, 10x concentrated mother liquor was prepared according to 1 / 4MS standard (Murashige & Skoog, 1962), with the KNO3 content doubled to 3.8 g / L, the dissolution process was carried out at a constant temperature of 40±2℃, and the crystallization completely disappeared by stirring at 300 rpm for 30 min, and then filtered and dispensed into amber glass bottles, stored at 4℃ in the dark to obtain the mother liquor;

[0037] S2, active ingredient nanofication treatment: Dissolve 5 mg of ATP-Na2 and 0.05 mg of shikonin in 10 mL of ultrapure water, under the condition of ice bath maintaining at 4℃, ultrasonic treatment at 200W power and 40kHz frequency for 15 min to form a preliminary complex; then add 0.01% polylactic acid-hydroxyacetic acid copolymer, and stir at 150 rpm for 2 h to obtain ATP-shikonin nanomicelles;

[0038] S3, culture solution mixing and sterilization: Take 800 mL of ultrapure water, add 100 mL of 1 / 4 mother liquor, 90 g of sucrose, 500 mg of hydrolyzed casein and 2 g of trehalose in sequence, stir at 400 rpm in a constant temperature water bath at 45℃ for 15 min until completely dissolved, cool to 30℃, then add ATP-shikonin nanomicelles and 0.1 mg of paclobutrazol dissolved in 0.5 mL of 70% ethanol, reduce the stirring speed to 100 rpm to avoid micelle shear damage, adjust the pH to 6.0±0.05 with 0.1M KOH / HCl, and then pour into pressure-resistant PETG culture bottles, sterilize at 0.12 MPa, 121℃ for 15 min to obtain the optimized culture solution.

[0039] The variety of the tuber in the tuber method in the above-mentioned examples is Huashu No. 18, and the potato stem segment is a single-node stem segment of a detoxification test tube seedling, with a length of 0.5-1.0 cm.

[0040] Comparative Example 1:

[0041] The difference from Example 1 is that no liquid medium is added, and the rest is consistent with Example 1.

[0042] Comparative Example 2:

[0043] The difference from Example 1 is that the short-day mode is not switched, and the initial culture stage conditions are used throughout, and the rest is consistent with Example 1.

[0044] Comparative Example 3:

[0045] The difference from Example 1 is that there is no blue light and red light pulse irradiation, and the synergistic regulation of blue light (400-500 nm) and red light (660 nm) is missing, and the rest is consistent with Example 1.

[0046] Comparative Example 4:

[0047] The difference from Example 1 is that the liquid medium is added: 1 / 4MS+6.0% sucrose is used to prepare pH6.2 liquid medium, and 25ml liquid medium is added to the culture container after the initial culture stage, and the rest is consistent with Example 1.

[0048] Comparative Example 5:

[0049] The difference from Example 1 is that the liquid medium is added: 1 / 4MS+9.0% sucrose is used to prepare pH6.2 liquid medium, and 15ml liquid medium is added to the culture container after the initial culture stage, and the rest is consistent with Example 1.

[0050] Comparative Example 6:

[0051] The difference from Example 1 is that the liquid medium is added: 1 / 4MS+6.0% sucrose is used to prepare pH6.2 liquid medium, and 15ml liquid medium is added to the culture container after the initial culture stage, and the rest is consistent with Example 1.

[0052] Performance test:

[0053] I. The test of tuber deformity rate and moldy rate during storage was carried out on the tubers planted by the method of producing indoor seed tubers of examples and comparative examples. The tuber deformity was defined as the standard of "Processing Potatoes" (NY / T 1605-2008). The external defects included: bifurcation, cracking, green skin, mechanical damage with depth > 2 mm, and the internal defects included: hollow diameter > 5 mm, black heart, necrotic spot. The re-inspection was screened by artificial vision. The formula was: deformity rate = (number of deformed tubers / total number of samples) x 100%. The moldy rate during storage was tested under the conditions of temperature 4 ± 0.5 ℃, relative humidity 85-90%, and lightless storage. The moldy determination method included the following steps: first, sensory detection, i.e. the surface appeared white mycelium, black mold or soft rot was judged as moldy; then, biochemical verification: 50 tubers were randomly taken every 20 days, and the volatile marker 1-octen-3-ol was detected. The content > 0.1 μg / g was judged as moldy. The formula was: moldy rate = (number of moldy tubers / total number of samples) x 100%. The test results were shown in Table 1.

[0054] Table 1:

[0055]

[0056] As shown in Table 1, the deformity rate and moldy rate of Example 1 were lower than those of comparative examples. Short day was the core condition for inducing tuber differentiation. The combination of blue light / red light had synergistic effect on tuber morphological establishment and stress resistance. Insufficient sucrose concentration (comparative example 4) or insufficient liquid volume (comparative example 5) would lead to increased moldy rate. Insufficient osmotic pressure affected tuber dry matter accumulation. When both of them were insufficient (comparative example 6), the moldy rate was the highest (42%), which verified that "9% sucrose + 25 ml liquid" of the example was the optimal ratio.

[0057] II. The number and size of harvested potatoes were tested. Each example and comparative example had 50 culture containers. The number and size of potatoes harvested from 50 containers were used as evaluation criteria. The result set was shown in Table 2.

[0058] Table 2

[0059]

[0060] The test results showed that the seed yield was the highest in Example 1, and the proportion of large weight seeds was the highest. With the increase of sucrose concentration and the volume of culture liquid, the seed yield and the proportion of large weight also increased. The best tuber formation condition was observed under the sucrose concentration of 9%. The culture liquid showed the best production volume at 25 ml. The liquid was absorbed within 30-40 days after dispensing at 15 ml, and the tuber growth was slow in the later stage.

[0061] It will be apparent to those skilled in the art that various modifications and variations can be made to the present application without departing from the spirit or scope of the application. Such modifications and variations are intended to fall within the scope of the application as defined by the appended claims and their equivalents.

Claims

1. A method for producing indoor seed potatoes using solid and liquid culture media, characterized in that, Includes the following steps: S1. Initial culture stage: Potato stem segments were inoculated into a solid culture medium and cultured for 25 days; S2. Liquid culture medium addition: Add 25 ml of liquid culture medium; S3. Late-stage culture: Culture for 80 days under 4 hours of light and 20 hours of darkness, with humidity maintained at 85–90%; The liquid culture medium was 1 / 4 MS basal medium with 9.0% sucrose added and a pH of 6.

2. The later cultivation stage also includes light regulation, which includes 4 hours of 400–500 nm blue light per day as the main light source; The later stages of cultivation also include light regulation, which includes 660 nm red light pulses 1–3 times a day for 15–25 minutes each time as an auxiliary light source.

2. The method according to claim 1, characterized in that, Potato stem segments were inoculated into solid culture medium and cultured for 25 days under conditions of 24±1℃, 4000 lux light intensity, 16 hours of light and 8 hours of darkness.

3. The method according to claim 1, characterized in that, The solid culture medium was 1 / 2MS basal medium, with 3.0% sucrose and 10 g / L agar added, and the pH value was 5.

7.

4. The method according to claim 1, characterized in that, The solid culture medium was placed in a culture container with dimensions of 7 cm × 7 cm × 10 cm, and 6 stem segments were inoculated in each culture container.

5. The method according to claim 1, characterized in that, The potato stem segments were single-segment stem segments of virus-free test-tube seedlings, with a length of 0.5–1.0 cm.

6. The method according to claim 1, characterized in that, The later culture stage also includes injecting 25 ml of optimized culture medium into the bottom of the culture container on the first day of the later culture stage. The injection position is the pre-reserved injection hole on the side wall of the container. The injection needle is inserted at a 45° angle and the flow rate is controlled at 2 ml / min. After injection, let it stand for 10 minutes and shake to promote the uniform diffusion of the culture medium into the solid culture medium matrix.

7. The method according to claim 6, characterized in that, The optimized culture medium comprises the following components: 1 / 4MS stock solution, 90 g / L sucrose, 500 mg / L hydrolyzed casein, 2 g / L trehalose, 0.01% polylactic acid-glycolic acid copolymer-encapsulated ATP-shikonin nanomicelles, and 0.1 mg / L paclobutrazol; the pH of the optimized culture medium is 6.0 ± 0.05; the 1 / 4MS stock solution is prepared by doubling the KNO3 content to 3.8 g / L, dissolving and stirring at 40 ± 2 °C for 30 minutes, filtering, and storing at 4 °C protected from light; the ATP-shikonin nanomicelles are prepared by the following steps: 5 mg ATP-Na2 and 0.05 mg shikonin are dissolved in 10 mL of ultrapure water, sonicated at 200 W power and 40 kHz frequency for 15 minutes in an ice bath at 4 °C, and then 0.01% polylactic acid-glycolic acid copolymer is added and stirred at 150 rpm for 2 hours.

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