Method for establishing sweet potato suspension cell line based on visual reporter gene

Abstract

The application discloses a sweet potato suspension cell line establishment method based on a visual reporter gene, and comprises the following steps: S1, taking a sweet potato tissue culture seedling as a material source, cutting a stem section, immersing the stem section in a resuspended solution of Agrobacterium rhizogenes carrying a RUBY expression vector to perform infection and then performing co-culture; after washing with a sterile aqueous solution of cefotaxime sodium, the stem section is inoculated into a rooting culture medium to perform hairy root induction culture and obtain red transgenic positive roots; S2, taking the red transgenic positive roots as explants, cutting the explants into sections, and inoculating the sections into a callus induction culture medium to perform callus induction culture; S3, inoculating the callus into a liquid culture medium to perform suspension cell start-up culture and subculture; and S4, culturing the suspension cells obtained in S3 in the callus induction culture medium in S2, and repeating S3. The application applies a visual reporter gene system to establishment of a sweet potato transgenic suspension cell line, and realizes visual screening of a whole cycle from a hairy root, a callus to a suspension cell.

Description

Technical Field

[0001] This invention relates to the field of plant biotechnology, specifically to a method for establishing a sweet potato suspension cell line based on a visual reporter gene. Background Technology

[0002] Plant cell culture technology, as an important branch of modern biotechnology, has demonstrated enormous application potential in fields such as plant secondary metabolite production, variety improvement, and genetic engineering research. Among these, suspension cell culture technology, due to its significant advantages such as good cell dispersibility, rapid growth rate, high proliferation coefficient, and ease of large-scale scaling, has become one of the core technologies for plant cell engineering research and industrial applications. Compared to traditional solid culture systems, suspension cell culture can achieve efficient transfer of nutrients, gases, and metabolites through a dynamic liquid environment, significantly improving the uniformity and stability of cell populations, and providing key technical support for the construction of standardized experimental systems and industrial production.

[0003] sweet potato( Ipomoea batatas (L.) Lam., a herbaceous plant of the Convolvulaceae family, is a globally important root crop used for food, feed, industrial raw materials, and new energy sources (starch and alcohol). However, sweet potato is an allohexaploid crop with a complex genome structure, diverse genetic background, and large differences in explant regeneration capacity, which poses significant challenges to genetic transformation and the establishment of suspension cell lines.

[0004] Traditional suspension culture methods mostly rely on shoot tip explants to induce embryogenic callus, and then establish suspension cell lines from the embryogenic callus. This method has the following prominent problems: limited explant sources, with shoot tip sources being limited and difficult to remove; strong genotype dependence, requiring 6-8 weeks to induce embryogenic callus on media containing 2,4-D, with some varieties being difficult to induce or having extremely low induction rates; unstable transformation efficiency, with large fluctuations and poor reproducibility in transformation efficiency using embryogenic callus as the recipient; long overall cycle and high cost, typically requiring 3-4 months from explant to the establishment of a suspension cell line, making it difficult to meet the needs of rapid research and development; furthermore, the plant regeneration cycle through suspension cells is long, and the proportion of deformed seedlings is relatively high.

[0005] Currently, sweet potato genetic transformation mainly employs Agrobacterium rhizogenes-mediated methods or Agrobacterium tumefaciens-mediated methods, with selection markers typically relying on antibiotic resistance genes (such as kanamycin and hygromycin) or fluorescent reporter genes (such as GFP). However, this approach suffers from drawbacks such as long selection cycles (weeks to months), high false-positive rates, cytotoxicity, reliance on specialized equipment like fluorescence microscopes, and biosafety risks. In recent years, the RUBY reporter gene system, based on the betaine synthesis pathway, has been developed, enabling transgenic tissues to produce a visible red pigment. It offers advantages such as no substrate required, no special equipment needed, and no cytotoxicity, and has been successfully applied in crops like Arabidopsis thaliana, rice, and tomato. However, the application of the RUBY system to Agrobacterium rhizogenes-mediated genetic transformation and suspension cell line establishment in sweet potato has not yet been systematically reported. Specifically, there is a lack of systematic research on utilizing the RUBY red phenotype to achieve full-process visual monitoring from transgenic hairy root culture to callus formation and transgenic cell suspension culture, and on establishing a rapid, efficient, and universally applicable method for sweet potato transgenic suspension cell lines that does not rely on antibiotics. Summary of the Invention

[0006] The technical problem to be solved by this invention is how to establish a transgenic sweet potato suspension cell line.

[0007] The present invention solves the above-mentioned technical problems through the following technical means:

[0008] A method for establishing a sweet potato suspension cell line based on a visual reporter gene includes the following steps: S1. Using sweet potato tissue culture seedlings as material, stem segments were cut as explants and immersed in Agrobacterium rhizogenes resuspension carrying the RUBY expression vector for infection. Then, the explants were removed, the surface bacterial solution was dried, and they were transferred to co-culture medium for culture. Afterward, they were washed with sterile aqueous solution of cefotaxime sodium and inoculated into rooting medium for hairy root induction culture to obtain red transgenic positive roots. S2. Using the red transgenic positive roots obtained in S1 as explants, the roots were cut into segments and inoculated into callus induction medium for callus induction culture. S3. The obtained callus tissue was inoculated into liquid culture medium for suspension cell initiation culture; the obtained cells were then subcultured on liquid culture medium. S4. Culture the suspended cells obtained in S3 in the callus induction medium in S2 to obtain callus tissue, and repeat step S3.

[0009] In this invention, by performing steps S1 to S3, a transgenic sweet potato suspension cell line can be established for the first time, which can be used for basic gene manipulation and short-term experiments. However, long-term passage in shake flasks of liquid suspension is prone to degeneration problems such as cell cluster browning, loss of embryonogenesis, decline in proliferation and viability, silencing of the target marker gene (red fading), and enrichment of trace Agrobacterium residues. After step S4, the suspension cells are returned to solid callus medium for rejuvenation: the growth rate is slowed down in the solid environment, the cell differentiation state is reset, and new callus without browning and with a stable red phenotype is selected. Then, the suspension is restarted to achieve cell line purification, viability rejuvenation, and long-term seed preservation and subculturing.

[0010] Preferably, in S1, the sweet potato is "Pu32".

[0011] Preferably, in S1, the Agrobacterium rhizogenes resuspension is prepared by resuspending Agrobacterium rhizogenes in a liquid culture medium, wherein the liquid culture medium is MS liquid culture medium with 100 μmol / L acetylsylgenone and 30 g / L sucrose added, and pH= 5.8-6.0.

[0012] Preferably, in S1, the co-culture medium is MS medium supplemented with 100 μmol / L acetylsalicylic acid, 6.8 g / L agar and 30 g / L sucrose, with a pH of 5.8-6.0.

[0013] Preferably, in S1, the rooting medium is MS medium supplemented with 300 mg / L cephalosporin, 6.8 g / L agar and 30 g / L sucrose, with a pH of 5.8-6.0.

[0014] Preferably, in S2, the callus induction medium is MS medium supplemented with 2.0 mg / L cyproconazole, 300 mg / L cephalosporin, 6.8 g / L agar, and 30 g / L sucrose, and the pH of the medium is 5.8-6.0.

[0015] Preferably, in S3, the liquid culture medium is MS medium containing 1-2.0 mg / L cyproconazole, 300 mg / L cephalosporin, and 10-50 g / L sucrose, with a pH of 5.8-6.0; the sucrose concentration can be 10, 20, 30, 40, or 50 g / L.

[0016] Preferably, in S3, the liquid culture medium is MS medium containing 2.0 mg / L cyproconazole, 300 mg / L cephalosporin, and 30 g / L sucrose, with a pH of 5.8-6.0.

[0017] Preferably, in S1, a 1-3 cm stem segment is cut as an explant; and / or, the Agrobacterium rhizogenes is Agrobacterium rhizogenes K599; and / or, the culture time on the co-culture medium is 2-3 days; and / or, the concentration of cefotaxime sodium in the sterile aqueous solution of cefotaxime sodium is 500 mg / L; and / or, the hairy root induction culture time is 7-14 days.

[0018] Preferably, in S2, the cells are cut into segments of 0.5-1.0 cm in size and inoculated into callus induction medium for callus induction culture; and / or, in S3, 0.05-0.3 g of callus tissue is inoculated into 10 mL of liquid culture medium for initiation culture of suspension cells; and / or, the initiation culture time is 2 weeks; and / or, 0.05-0.3 g of fresh cell weight is suspended in 10 mL of liquid culture medium for subculture.

[0019] Preferably, suspension cell initiation culture is performed by inoculating 0.05g, 0.1g, 0.15g, 0.2g or 0.3g of callus tissue into 10 mL of liquid culture medium.

[0020] Preferably, subculture is performed by suspending 0.05g, 0.1g, 0.15g, 0.2g or 0.3g of fresh cell weight in 10 mL of liquid culture medium.

[0021] Preferably, in S4, the culture time is 4 weeks; and / or, the culture conditions in S1 (hairy root induction culture), S2 (callus induction culture), and S4 (callus induction medium) are a temperature of 25 ± 2 ℃ and a light intensity of 40-50 μmol·m⁻¹. -2 s -1 The photocycle is 14h light / 10h dark; and / or, the conditions for initiating and subculturing the suspension cells described in S3 are: under dark conditions, constant temperature of 26℃, and shaking at 120 rpm.

[0022] Preferably, in S1, the infection time is 15-25 min; more preferably 20 min; the co-culture medium is used for dark culture.

[0023] Preferably, during the subculture process, subculture is performed every 2 weeks.

[0024] Preferably, in S3, during the subculture process, the culture is inoculated at a volume ratio of 1:10 for suspension culture and liquid culture medium.

[0025] The advantages of this invention are: This invention uses the RUBY betaine pathway as the screening marker. Transgenic tissues spontaneously turn red, allowing for direct visual identification of positive results. The entire screening process relies on the red phenotype, without using antibiotic resistance screening markers such as kanamycin or hygromycin. The addition of cephalosporin / cefotaxime sodium to the culture medium is solely for killing Agrobacterium tumefaciens, and positive results are not determined by antibiotic resistance. This invention applies a visual reporter gene system to the establishment of transgenic sweet potato suspension cell lines, achieving full-cycle visual screening from hairy roots and callus tissue to suspension cells, without requiring special equipment. It boasts high propagation efficiency, good genetic stability, and is easy to scale up, providing an efficient, stable, and visual technical platform for sweet potato biotechnology breeding, gene function research, and the industrialization of cell engineering. Attached Figure Description

[0026] Figure 1 This is a map of the RUBY expression vector plasmid in Example 1 of the present invention; Figure 2 These are the positive hairy roots produced on the transformed sweet potato seedling segments in Example 1 of this invention; Figure 3 The root segment inoculated on the callus induction culture medium in Example 1 of this invention; Figure 4 This refers to the callus tissue induced by positive hairy roots after 3 weeks of culture in Example 1 of this invention. Figure 5 This is the state of the positive callus tissue after 2 weeks of liquid culture in Example 1 of the present invention; Figure 6 This refers to the renewal culture of the suspension culture inoculated onto the callus induction medium in Example 1 of the present invention; Figure 7 The transgenic suspension cell hygromycin resistance gene in Example 1 of this invention hygR PCR electrophoresis detection image; Figure 8 This shows the growth status of sweet potato suspension culture cells under different PGRs combinations in Example 3 of the present invention; Figure 9 This is the growth curve of sweet potato suspension culture cells in Example 5 of the present invention. Detailed Implementation

[0027] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions of the embodiments of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0028] Unless otherwise specified, all test materials and reagents used in the following examples are commercially available.

[0029] Unless otherwise specified in the embodiments, the techniques or conditions described in the literature in this field or in accordance with the product manual may be followed.

[0030] The following are the methods for propagating sweet potato tissue culture seedlings: Select healthy, disease-free Pu 32 seed potatoes, wash and dry them with clean water, then germinate them at 25℃ in the dark and moisturized environment. Once axillary buds have sprouted, cut the bud strips. Rinse with running water for 30 min, then sterilize in a clean bench with 75% (v / v) ethanol for 30 s, followed by 0.1% (w / v) HgCl2 with shaking for 6 min. Rinse five times with sterile water to remove residual disinfectant. After blotting with sterile filter paper, cut into single-segment stem sections and inoculate onto MS basal solid medium at pH 5.8–6.0, supplemented with 30 g / L sucrose and 6.8 g / L agar. Induce seedlings through primary culture at 25±2℃ under a 14 h light / 10 h dark photoperiod. Subsequently, cut single-segment stem sections and inoculate them onto MS basal solid medium at pH 5.8–6.0, supplemented with 30 g / L sucrose and 6.8 g / L agar, supplemented with 2 mg / L HgCl2. Meta-Topolin was subcultured on MS basal solid medium, with subculture every 30 days. After 4-6 weeks of propagation culture, sterile virus-free tissue culture seedlings with strong stems and uniform growth were obtained. 1-3 cm stem segments were cut as explant materials for subsequent Agrobacterium rhizogenes genetic transformation.

[0031] Example 1 A method for establishing a sweet potato suspension cell line based on a visualized reporter gene is described below: (1) Establishment of the Agrobacterium rhizogenes-mediated sweet potato genetic transformation system: Constructing a vector carrying RUBY expression ( Figure 1 The specific steps for using Agrobacterium rhizogenes K599 are as follows: 100 μL of Agrobacterium rhizogenes K599 chemocompetent cells (purchased from Shanghai Weidi Biotechnology Co., Ltd.) were thawed in an ice bath. 0.5–1 μg of the RUBY recombinant plasmid, which had been verified by sequencing, was added. The mixture was gently thawed and then incubated in an ice bath for 5 min. The cells were then flash-frozen in liquid nitrogen for 5 min, heat-shocked in a 37°C water bath for 5 min, and then incubated in an ice bath for 5 min again. 700 μL of antibiotic-free TY liquid medium was added, and the cells were thawed at 28°C and 200 rpm for 2 h. The cells were centrifuged at 6000 rpm for 1 min, and part of the supernatant was discarded. The remaining cells were resuspended in antibiotic-free TY liquid medium and spread on TY solid plates containing 50 mg / L streptomycin + 50 mg / L spectinomycin. The plates were incubated upside down at 28°C for 2–3 days. Single colonies were picked for RUBY gene-specific colony PCR identification to screen for positive transformants. Positive single colonies were inoculated into TY liquid medium containing 50 mg / L streptomycin + 50 mg / L spectinomycin and activated overnight at 28°C and 180 rpm. Glycerol-containing bacteria were stored at -80°C. Preparation of infection solution: The activated bacterial solution was transferred to TY liquid medium containing 50 mg / L streptomycin + 50 mg / L spectinomycin and cultured at 28 °C with shaking until OD. 600 =0.8, centrifuge to collect bacterial cells, resuspend the cells in MS liquid medium (pH=5.8–6.0) supplemented with 100 μmol / L acetylsalicylic acid and 30 g / L sucrose, and adjust OD. 600 The concentration was increased to 0.4-0.6, and the mixture was activated by shaking at 28℃ and 180 rpm for 1-2 h to obtain Agrobacterium rhizogenes K599 infection solution; Sweet potato explant infection and hairy root induction: Tissue culture seedlings of approximately 6 weeks old sweet potato variety "Pu 32" were used as the material source. 1-3 cm segments were cut from the seedlings and used as explants. These segments were immersed in the *Agrobacterium rhizogenes* K599 infection solution carrying the RUBY expression vector for 20 min, with gentle shaking during the process. After removing the explants and drying the surface bacterial solution, they were transferred to MS co-culture medium (pH = 5.8-6.0) supplemented with 100 μmol / L acetylsylgenone, 6.8 g / L agar, and 30 g / L sucrose and cultured in the dark for 2 days. Then, the dark-cultured sweet potato stem segments were washed in sterile water supplemented with 500 mg / L cefotaxime sodium for approximately 15 min, dried on sterile filter paper, and inoculated into a solution supplemented with 300 mg / L cephalosporin, 6.8 g / L agar, and 30 g / L sucrose. Hairy root induction culture was carried out in MS rooting medium (pH=5.8-6.0) with g / L sucrose; the culture conditions were 25 ± 2 ℃ temperature and 40-50 μmol·m² light intensity. -2 s -1 In a greenhouse with a photoperiod of 14 / 10 h (light / dark), after 7 days of cultivation, visible red transgenic positive roots formed at the base of the stem segments. Figure 2 ).

[0032] (2) Induction of transgenic hairy root callus: After culturing for another week, the red positive roots selected in step (1) were used as explants and cut into segments of 0.5-1.0 cm in size. Figure 3 The callus tissue was inoculated into MS medium (pH 5.8-6.0) supplemented with 2.0 mg / L cyproconazole, 300 mg / L cephalosporin, 6.8 g / L agar, and 30 g / L sucrose and cultured in the greenhouse described in (1) above. After 3 weeks of culture, loose callus tissue was obtained. Figure 4 ).

[0033] (3) Establishment of a positive transgenic callus suspension culture system: Take the fragile granular positive callus obtained in step (2), inoculate 1 g of callus into 50 mL of liquid culture medium, and start suspension culture at 120 rpm and 26℃ in the dark; after 2 weeks of culture ( Figure 5 The callus tissue was filtered through a 500 μm mesh to remove clumped tissue, and then red suspended cells were collected through a 50 μm sterile nylon mesh. The cells were then suspended at 1 g fresh weight in 50 mL of liquid culture medium and subcultured under the same conditions of 26 ℃ and 120 rpm in the dark with shaking. The liquid culture medium was MS medium supplemented with 2.0 mg / L cyproconazole, 300 mg / L cephalosporin, and 30 g / L sucrose, with a pH of 5.8-6.0.

[0034] (4) Proliferation culture of transgenic suspension cells: Take the sweet potato transgenic positive suspension cells obtained after subculture in step (3), take 5 mL of suspension culture and inoculate it into 50 mL of liquid culture medium in (3), culture in the dark at 26℃ and 120 rpm, and subculture once every 2 weeks; (5) Renewal culture of transgenic suspension cells: Take the sweet potato suspension cells obtained in step (4), use a sterile pipette tip to aspirate 1-2 mL of suspension culture onto the culture medium in step (2), use a spreading stick to evenly spread the suspension cells onto the surface of the culture medium in step (2), tilt the culture medium to remove excess culture medium or blow off excess moisture in a clean bench; after culturing in the greenhouse in step (1) for 4 weeks ( Figure 6 Select healthy callus tissue that is still red and not browned, and repeat steps (3) and (4).

[0035] (6) Molecular identification of transgenic suspension cells (PCR verification of hygromycin resistance gene) Four types of materials were collected: ① transgenic sweet potato suspension cells that had undergone multiple subcultures (more than 2 subcultures) and were stably red; ② untransformed sweet potato suspension cells (negative cell control); ③ sterile deionized water (blank control); ④ recombinant plasmids carrying the RUBY reporter gene (positive control); at the same time, the suspension culture was spread on LB solid plates and incubated at 28°C for 3 days. No Agrobacterium colonies grew, thus eliminating interference from Agrobacterium cell pigments.

[0036] Genomic DNA extraction: Red sweet potato transgenic suspension cells and untransformed sweet potato cells were thoroughly ground with liquid nitrogen and total DNA was extracted using a plant genomic DNA extraction kit. The purity and concentration of DNA were detected by Nanodrop and diluted to 50 ng / μL for later use.

[0037] Hygromycin resistance gene hygR Specific PCR amplification: Specific upstream and downstream primers were designed based on the hygromycin resistance gene sequence carried by the vector, as shown in Table 1. The reaction system (25 μL) included: 2×PCR Mix: 12.5 μL; upstream primer (10 μmol / L): 1.0 μL; downstream primer (10 μmol / L): 1.0 μL; template DNA (50 ng / μL): 2.0 μL; ddH2O to a final volume of 25 μL. The amplification program was as follows: 94 ℃ pre-denaturation for 5 min; 94 ℃ denaturation for 45 s, 58 ℃ annealing for 45 s, 72 ℃ extension for 60 s, for a total of 35 cycles; final extension at 72 ℃ for 10 min; and storage at 4 ℃.

[0038] Electrophoresis detection: The amplification products were separated by 1.5% (w / v) agarose gel electrophoresis, stained with GreenView nucleic acid dye, and the bands were observed and recorded using a gel imaging system. The appearance of a specific target band was used to determine PCR positive transformation material.

[0039] Results: The red transgenic suspension cell sample amplified with... hygR Specific bands of the same size as the expected gene ( Figure 7 (As shown in 1-6); Untransformed sweet potato cells (negative) and sterile water blank control (NTC) showed no amplification bands, proving that the RUBY expression vector was fully integrated into the sweet potato suspension cell genome and belonged to stable transgenic cells. The red phenotype was not caused by transient expression or Agrobacterium contamination; M in the figure represents Marker, DNA molecular weight standard.

[0040] Table 1 hygR Gene PCR detection primer sequences

[0041] Example 2 This embodiment tested the effect of sucrose concentration on the suspension culture of sweet potato transgenic cells. The specific steps are as follows: Sweet potato suspension cells in the logarithmic growth phase obtained from step (4) of Example 1 for 2 weeks were taken and filtered through a sterile filter with a pore size of 500 μm to remove the clumps of callus tissue in the system; then, single cells and small cell clusters were collected by intercepting them through a sterile nylon mesh with a pore size of 50 μm. The collected cells were inoculated into 10 mL of liquid culture medium containing different sucrose concentrations at a cell weight of 0.1 g fresh weight. The sucrose concentration gradient was set to 10, 20, 30, 40, and 50 g / L. Except for the sucrose concentration, the other components and concentrations of the culture medium were kept consistent with the culture medium in step (3) of Example 1. Three biological replicates were set for each treatment. The inoculated culture medium was placed in a constant temperature shaker and cultured in the dark at a speed of 120 rpm and a temperature of 26℃ for 2 weeks. After the culture period, the fresh weight and dry weight of the suspension cells in each group were measured (the dry weight measurement required drying at 60℃ to constant weight before weighing). The results are shown in Table 2. The data indicate that sucrose concentration significantly affected the fresh weight and dry weight of sweet potato suspension cells. p <0.05). Fresh weight increased significantly with increasing sucrose concentration from 10 to 30 g / L, reaching its highest value (123.84 ± 5.6 g / L, a) at 30 g / L, and decreased after exceeding 30 g / L. Dry weight remained at a high level at concentrations of 30 g / L and above, significantly higher than the 10–20 g / L group. 30 g / L sucrose can balance carbon source supply and osmotic pressure, providing optimal conditions for cell growth; low concentrations of insufficient carbon source inhibit growth, while high concentrations hinder fresh weight growth due to excessively high osmotic pressure. Considering both indicators, the optimal sucrose concentration was determined to be 30 g / L.

[0042] Table 2. Effects of different sucrose concentrations on the growth of sweet potato suspension culture cells.

[0043] Note: Different lowercase letters after the data in the same column indicate significant differences. p <0.05), data are the mean ± standard deviation of 3 biological replicates.

[0044] Example 3 This embodiment tested the effects of different types and concentration combinations of plant growth regulators (PGRs) on the growth characteristics of sweet potato suspension culture cells. The specific steps are as follows: Sweet potato suspension cells in the logarithmic growth phase obtained after 2 weeks of culture in step (4) of Example 1 were taken and filtered through a sterile filter with a pore size of 500 μm to remove clumps of callus tissue from the system; then, single cells and small cell clusters were collected by retaining them through a sterile nylon mesh with a pore size of 50 μm. The collected cells were inoculated into 10 mL of liquid culture medium at a fresh weight of 0.05 g cells. The plant growth regulators in the culture medium were prepared as follows: Picloram 1 mg / L (P1), Picloram 2 mg / L (P2), Picloram 2 mg / L + meta-Topolin 0.5 mg / L (PM), 2,4-D 2 mg / L (D2), and 2,4-D 2 mg / L + 6-BA 0.2 mg / L (DB). Except for the plant growth regulators, the other components and concentrations of the culture medium were kept consistent (MS medium with 300 mg / L cephalosporin and 30 g / L sucrose, pH 5.8-6.0). Each treatment was performed in triplicate. The inoculated culture medium was placed in a constant temperature shaker and cultured in the dark at 120 rpm and 26℃ for 2 weeks. After the culture period, the fresh weight and dry weight of the suspended cells in each group were measured. The results are shown in Table 3. The experimental data show that different types and concentrations of PGRs significantly affected the fresh and dry weight growth of sweet potato suspension culture cells (P<0.05): There was no significant difference in fresh and dry weight between groups P1 and P2, and P1 was significantly higher than the other treatment groups; while the fresh and dry weights of groups PM, D2, and DB were significantly lower, with no significant differences among the three groups. From a biological perspective, metatopolin promotes the proliferation and accumulation of substances in sweet potato suspension cells, while the combination of metatopolin and metatopolin, 2,4-D alone, and the combination of 2,4-D and 6-BA are detrimental to cell growth. Figure 8 Based on both fresh weight and dry weight, the optimal PGRs conditions for sweet potato suspension culture cells were determined to be 2 mg / L atrazine (no significant difference compared to 1 mg / L, but cell growth was slightly better under the 2 mg / L treatment). Table 3. Effects of different PGR types and concentration combinations on the growth of sweet potato suspension culture cells.

[0045] Note: Different lowercase letters after the data in the same column indicate significant differences (p<0.05). The data are the mean ± standard deviation of three biological replicates.

[0046] Example 4 This embodiment tested the effect of inoculum size on the growth of suspension culture. The specific steps are as follows: Sweet potato suspension cells in the logarithmic growth phase obtained from step (4) of Example 1 after 2 weeks of culture were taken and filtered through a sterile filter with a pore size of 500 μm to remove the callus tissue in the system; then, single cells and small cell clusters were collected by intercepting them through a sterile nylon mesh with a pore size of 50 μm. Using fresh weight as the measurement standard, the collected cells were inoculated into 10 mL of liquid culture medium from step (3) of Example 1 according to different inoculum size gradients. The inoculum size gradients were set to 0.05, 0.1, 0.15, 0.2, and 0.3 g / 10 mL, and each treatment was set up with 3 biological replicates. The inoculated culture medium was placed in a constant temperature shaker and cultured in the dark at a speed of 120 rpm and a temperature of 26℃ for 2 weeks; after the culture period, the fresh weight and dry weight of the suspension cells in each group were measured. The results are shown in Table 4. The experimental data show that the inoculum size has a highly significant effect on the growth of both fresh weight and dry weight of sweet potato suspension culture cells. p <0.01), and the overall trend shows a significant increase with increasing inoculum amount. The inoculum amount directly determines the initial cell density. At low inoculum amounts (0.05~0.1 g / 10 mL), the cell density is too low, resulting in insufficient intercellular signal transduction, low nutrient utilization efficiency, and slow cell proliferation. With increasing inoculum amount, the initial cell density is suitable, the intercellular synergy is enhanced, nutrients are utilized efficiently, and the cell growth rate is accelerated. Considering both fresh weight and dry weight indicators, within the inoculum amount gradient range set in this example, the sweet potato suspension cell growth state of the 0.3 g / 10 mL treatment group is the best. However, considering the actual culture cost and subsequent growth stability, an inoculum amount of 0.2 g / 10 mL is sufficient to meet the requirements for good cell growth and can avoid problems such as intense nutrient competition and accumulation of metabolites that may be caused by excessively high inoculum amounts. Therefore, the optimal inoculum amount is determined to be 0.2 g / 10 mL.

[0047] Table 4. Effects of different inoculum sizes on the growth of sweet potato suspension culture cells.

[0048] Note: Different lowercase letters after the data in the same column indicate significant differences. p <0.05), data are the mean ± standard deviation of 3 biological replicates.

[0049] Example 5 This example illustrates the growth curve of a transgenic sweet potato cell suspension culture. The specific steps are as follows: (1) Cell pretreatment: Sweet potato suspension cells in the logarithmic growth phase obtained by culturing for 2 weeks in step (4) of Example 1 were filtered through a sterile filter with a pore size of 500 μm to remove the clumps of callus tissue in the system; then single cells and small cell clusters were intercepted and collected through a sterile nylon mesh with a pore size of 50 μm for later use.

[0050] (2) Inoculation and culture: The pretreated cells were inoculated into 100 mL of liquid culture medium containing fixed components (i.e., the liquid culture medium in step (3) of Example 1) at an inoculation amount of 2 g / 100 mL fresh weight. Three biological replicates were set for each treatment. The inoculated culture medium was placed in a constant temperature shaker and cultured in the dark at a speed of 120 rpm and a temperature of 26°C for 24 days.

[0051] (3) Sampling and Measurement: Starting from the day of inoculation (day 0), samples were taken at fixed time intervals on days 0, 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, and 24. Each time samples were taken, three replicate samples from each group were taken, and the fresh weight, dry weight, packed cell volume (PCV), and electrical conductivity (EC) of the suspension culture medium in each bottle were measured. The dry weight was measured after drying at 60°C to constant weight. The cell volume was measured by centrifugation (centrifugation at 4000 rpm for 10 min, and the percentage of cell pellet volume to total culture medium volume was read). The electrical conductivity of the suspension culture medium was measured by a conductivity meter.

[0052] (4) Growth curve plotting: The growth curve of sweet potato suspension culture cells was plotted using GraphPad Prism software with culture time (days) as the x-axis and the mean ± standard deviation of fresh weight and dry weight of each group of cells as the y-axis. Figure 9 The study analyzed key stages of cell growth, including the lag phase, logarithmic growth phase, and stationary phase. Experimental data are shown in Table 5. Combined with growth curve data (time: 0–24 days; indicators: fresh weight, dry weight, PCV, EC), the growth cycle of sweet potato suspension culture cells can be divided into four stages: lag phase, logarithmic growth phase, stationary phase, and decline phase. The characteristics and indicator changes of each stage are as follows: ① Delayed phase (0-4 days): During this phase, cell fresh weight, dry weight, and PCV increase slowly, while EC decreases slowly. Cells are newly inoculated into the new culture medium and need to adapt to the environment and initiate metabolic regulation. At this time, cell proliferation rate is low, and the absorption of nutrients (such as ions) from the culture medium is minimal. Therefore, EC decreases gradually, and cell biomass accumulation is slow.

[0053] ② Logarithmic growth phase (4-16 days): During this phase, cells enter a rapid proliferation phase, with all growth indicators showing a significant upward trend and the growth rate reaching its peak. Simultaneously, the endocrine volume (EC) shows a rapid downward trend. Cellular metabolic activity is vigorous during this phase, with cells proliferating rapidly and efficiently absorbing nutrients (such as sucrose and mineral ions) from the culture medium, leading to a continuous decrease in EC. The number of cells increases rapidly, and biomass (fresh weight, dry weight) and cell volume (PCV) significantly increase, making this the core stage of cell proliferation.

[0054] ③ Stationary phase (16-18 days): After cell growth slows down and reaches its peak, it tends to stabilize. Fresh weight, dry weight, and PCV all remain at high levels. The growth peak occurs at day 16, and all indicators decrease slightly at day 18. EC drops to its lowest value and then slightly rebounds. During this stage, the cell proliferation rate and death rate are basically in balance. Nutrients in the culture medium are gradually depleted, and secondary products of cell metabolism begin to accumulate, leading to a slight rebound in EC. Cell biomass no longer increases significantly.

[0055] ④ Decline phase (18-24 days): All growth indicators show a continuous downward trend, while EC continues to rise. The reason is that the nutrients in the culture medium are completely depleted, and a large amount of metabolic products accumulate and produce toxicity, leading to a large number of cell deaths and a reduction in biomass; the dead cells release intracellular ions, causing the EC of the suspension culture medium to rise continuously.

[0056] Table 5. Effects of different culture times on growth parameters of sweet potato suspension culture cells.

[0057] Note: Data are the mean ± standard deviation of three biological replicates.

[0058] This invention discloses a method for establishing sweet potato suspension cell lines based on a visual reporter gene. Using stem segments of sweet potato 'Pu 32' tissue culture seedlings as explants, the stems are transformed with Agrobacterium rhizogenes-mediated transformation into expression vectors carrying visual reporter genes, resulting in stable, red, transgenic, positive hairy roots. These positive roots are then segmented and inoculated into callus induction medium to induce the formation of red, granular callus tissue. Fragile callus tissue is collected for liquid suspension culture, and the dispersed suspension cells are collected after filtration. Subculture and proliferation yield a transgenic suspension cell line. The suspension cells are then refreshed by spreading them onto a solid callus induction medium to maintain long-term cell viability. This invention applies a visual reporter gene system to the establishment of transgenic sweet potato suspension cell lines, achieving full-cycle visual screening from hairy roots and callus tissue to suspension cells, without requiring special equipment. It boasts high propagation efficiency, good genetic stability, and is easily scaled up, providing an efficient, stable, and visual technical platform for sweet potato biotechnology breeding, gene function research, and the industrialization of cell engineering.

[0059] This invention develops a visualized, efficient, and low-cost method for sweet potato genetic transformation and the establishment of transgenic suspension cell lines based on the RUBY reporter gene. This method has significant theoretical and practical implications for overcoming barriers in sweet potato biotechnology breeding and promoting its industrial application in cell engineering.

[0060] The above embodiments are only used to illustrate the technical solutions of the present invention, and are not intended to limit it. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for establishing a sweet potato suspension cell line based on a visual reporter gene, characterized in that: Includes the following steps: S1. Using sweet potato tissue culture seedlings as material, stem segments were cut as explants and immersed in Agrobacterium rhizogenes resuspension carrying the RUBY expression vector for infection. Then, the explants were removed, the surface bacterial solution was dried, and they were transferred to co-culture medium for culture. Afterward, they were washed with sterile aqueous solution of cefotaxime sodium and inoculated into rooting medium for hairy root induction culture to obtain red transgenic positive roots. S2. Using the red transgenic positive roots obtained in S1 as explants, the roots were cut into segments and inoculated into callus induction medium for callus induction culture. S3. The obtained callus tissue was inoculated into liquid culture medium for suspension cell initiation culture; the obtained cells were then subcultured on liquid culture medium. S4. Culture the suspended cells obtained in S3 in the callus induction medium in S2 to obtain callus tissue, and repeat step S3.

2. The method for establishing sweet potato suspension cell lines based on visual reporter genes according to claim 1, characterized in that: In S1, the sweet potato is "Pu32".

3. The method for establishing a sweet potato suspension cell line based on a visual reporter gene according to claim 1, characterized in that: In S1, the Agrobacterium rhizogenes resuspension is prepared by resuspending Agrobacterium rhizogenes in a liquid culture medium, wherein the liquid culture medium is MS liquid culture medium with 100 μmol / L acetosyringone and 30 g / L sucrose added, pH= 5.8-6.

0.

4. The method for establishing a sweet potato suspension cell line based on a visual reporter gene according to claim 1, characterized in that: In S1, the co-culture medium is MS medium supplemented with 100 μmol / L acetylsalicylic acid, 6.8 g / L agar and 30 g / L sucrose, with pH = 5.8-6.

0.

5. The method for establishing a sweet potato suspension cell line based on a visual reporter gene according to claim 1, characterized in that: In S1, the rooting medium is MS medium supplemented with 300 mg / L cephalosporin, 6.8 g / L agar and 30 g / L sucrose, with pH = 5.8-6.

0.

6. The method for establishing a sweet potato suspension cell line based on a visual reporter gene according to claim 1, characterized in that: In S2, the callus induction medium is MS medium supplemented with 2.0 mg / L cyproconazole, 300 mg / L cephalosporin, 6.8 g / L agar, and 30 g / L sucrose, with a pH of 5.8-6.

0.

7. The method for establishing a sweet potato suspension cell line based on a visual reporter gene according to claim 1, characterized in that: In S3, the liquid culture medium is MS medium containing 1-2.0 mg / L cyproconazole, 300 mg / L cephalosporin, and 10-50 g / L sucrose, with a pH of 5.8-6.

0.

8. The method for establishing a sweet potato suspension cell line based on a visual reporter gene according to claim 1, characterized in that: In S1, 1-3 cm stem segments are cut as explants; and / or, the Agrobacterium rhizogenes is Agrobacterium rhizogenes K599; and / or, the culture time on the co-culture medium is 2-3 days; and / or, the concentration of cefotaxime sodium in the sterile aqueous solution of cefotaxime sodium is 500 mg / L; and / or, the hairy root induction culture time is 7-14 days.

9. The method for establishing a sweet potato suspension cell line based on a visual reporter gene according to claim 1, characterized in that: In S2, the cells are cut into segments of 0.5-1.0 cm and inoculated into callus induction medium for callus induction culture; and / or, in S3, 0.05-0.3 g of callus tissue is inoculated into 10 mL of liquid culture medium for initiation culture of suspension cells; and / or, the initiation culture time is 2 weeks; and / or, 0.05-0.3 g of fresh cell weight is suspended in 10 mL of liquid culture medium for subculture.

10. The method for establishing a sweet potato suspension cell line based on a visual reporter gene according to any one of claims 1-9, characterized in that: In S4, the culture time is 4 weeks; and / or, the culture conditions for the hairy root induction culture in S1, the callus induction culture in S2, and the callus induction medium in S4 are a temperature of 25 ± 2 ℃ and a light intensity of 40-50 μmol·m⁻¹. -2 s -1 The photocycle is 14h light / 10h dark; and / or, the conditions for initiating and subculturing the suspension cells described in S3 are: under dark conditions, constant temperature of 26℃, and shaking at 120 rpm.

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