A method for permanent genetic transformation using embryonic callus of Pinus koraiensis

A method for permanent genetic transformation using embryonic callus of Pinus koraiensis | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article A method for permanent genetic transformation using embryonic callus of Pinus koraiensis Hui Hou, Hao Dong, Yanan Wu, Wenna Zhao, Hailong Shen, Ling Yang, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4173927/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Pinus koraiensis holds the designation of a second-class protected wild plant in China, valued for its significant economic and ecological importance. However, the lack of a well-established genetic transformation system has hindered progress in functional research and breeding applications for this species. This study aimed to establish a reliable and efficient genetic transformation system of Pinus koraiensis embryonic callus, building upon somatic embryogenesis technology. Utilizing Pinus koraiensis embryonic callus and GUS as the reporter gene, Agrobacterium-mediated transformation was employed to investigate key transformation factors, such as antibiotic type and concentration, Agrobacterium bacterial solution concentration, infiltration, and co-cultivation times. The results revealed that 10 mg·L-1 of Hygromycin (Hyg) significantly inhibited Pinus koraiensis embryonic callus proliferation, while an OD600 absorbance value of 0.6 during transformation led to a remarkable 93.42±2.13% efficiency. Optimal co-cultivation for two days resulted in a transformation rate of 82.61%, with a high GUS staining rate of 88.89% in the resistant embryonic callus. Following the optimized protocol, resistant somatic embryos were successfully obtained. This research contributes to the advancement of seed resource breeding and genetic enhancement for Pinus koraiensis, providing a solid foundation for investigating gene functions related to this species. Biological sciences/Biotechnology/Plant biotechnology Biological sciences/Genetics/Plant breeding Pinus koraiensis Embryogenic callus Agrobacterium-Mediated Genetic transformation Somatic Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Introduction Scientifically classified as an evergreen tree within the Pinus genus of the Pinaceae family, Pinus koraiensis , also known as the sea pine, is naturally distributed across China, particularly in the Great and Small Xing'an Mountains and the Changbai Mountains 1 . In northeastern China, Pinus koraiensis holds significant importance as a native timber species. Its significance extends beyond timber, as its sapwood, bark, pine needles, and cones serve as crucial industrial raw materials 2 . Aside from its industrial utility, Pinus koraiensis fruit is renowned for its rich nutritional content, making it a cornerstone of both the nut economy and timber forest sectors in China. Recognized for its ecological, economic, medicinal, and nutritional value 3 , Pinus koraiensis has been the subject of research focusing on genetic diversity, seedling reforestation, and resource utilization. Recent years have seen increased attention towards propagating and genetically enhancing Pinus koraiensis due to its substantial ecological and economic contributions. Despite facing challenges in widespread use for large-scale silvicultural seedling production due to its extended growth cycle and limited asexual propagation techniques, Pinus koraiensis shows promise in somatic embryogenesis. This technique, known for its stable heritability, rapid reproduction, and low mutation rate, offers potential for both the regeneration and genetic improvement of Pinus koraiensis . Researchers like Gao 4 , 5 have successfully generated Pinus koraiensis embryonic callus from immature seed embryos, leading to plant regeneration through somatic embryogenesis. Additionally, Peng et al. 6 - 9 utilized Pinus koraiensis embryonic callus to establish an ultra-low temperature preservation system and explored proliferation differences using transcriptome and metabolomics sequencing technology. These advancements refine crucial technological aspects of Pinus koraiensis somatic embryogenesis 10 . The Agrobacterium -mediated genetic transformation method has emerged as the most extensively studied, technically advanced, efficient, and widely utilized approach for genetic transformation in conifers 11 . Notably, Huang 12 pioneered the inoculation of sterile European larch seedlings with hairy Agrobacterium , leading to the successful production of transformed plants. Presently, transgenic embryogenic cultures have been effectively developed using the Agrobacterium -mediated technique in various tree species, including Japanese larch ( Larix kaempferi ), torch pine ( Pinus taeda ), and Norway spruce ( Picea abies ) 13 - 17 . By integrating the somatic embryogenesis pathway with genetic transformation technology 18 , the utilization of somatic embryogenesis enables the conversion of resistant callus tissues identified during genetic transformation into somatic embryos. These embryos can subsequently germinate to yield fully resistant plants, offering potentially higher efficiency compared to traditional organ explant transformation and generating purer transformed plants. In this study, Agrobacterium strain GV3101 was utilized along with the binary vector VB191103-1905rcy, employing Pinus koraiensis embryonic callus as the primary material. Through optimization of bacterial solution concentrations and infiltration times during the transformation process, resistant embryonic calli were successfully obtained. This established a straightforward and efficient Agrobacterium -mediated genetic transformation system for Pinus koraiensis embryonic tissue is poised to accelerate transgenesis development and enhance research into genetic improvement and molecular functions of Pinus koraiensis . Results Cef Effects on Embryonic Callus Cef did not significantly impact the inhibition of Pinus koraiensis embryonic callus (Table 1). With increasing Cef concentration, the proliferation rate of Pinus koraiensis embryonic callus gradually decreased after 15 days. At an additional concentration of 500 mg·L -1 , the fresh mass of the proliferating embryonic callus decreased by 10.01% compared to the control. The inhibitory effects on the embryonic callus of Pinus koraiensis were not significant across all tested Cef treatment concentrations (p > 0.05). Table 1. Different concentrations of Cef on the proliferation rate of embryonic callus Cef concentration (mg·L -1 ) Rate of Callus proliferation (%) 0 3.36±0.32 a 100 3.28±0.49 a 200 3.18±0.43 a 300 3.00±0.39 a 400 3.07±0.75 a 500 2.87±0.42 a Kan Effects on Embryonic Callus There were no significant differences in the proliferation rate of Pinus koraiensis embryonic callus with increasing Kan concentration (Figure 1). The proliferation ploidy of embryonic callus cultured for 15 days without Kan was 3.35. Upon adding Kan at concentrations of 10, 20, 40, 80, and 100 mg·L -1 , the proliferation rates were 2.89, 2.57, 2.59, 2.52, and 2.07, respectively. A significant difference (p < 0.05) in embryonic callus proliferation was observed only when the Kan concentration in the medium reached 100 mg·L -1 . However, no significant difference in the proliferation of Pinus koraiensis embryonic callus was noted when Kan concentration was below 100 mg·L -1 . Inhibitory effects were evident only at Kan concentrations exceeding 100 mg·L -1 . Hyg Effects on Embryonic Callus Hyg exerted a significant inhibitory effect on the proliferation of Pinus koraiensis embryonic callus. With increasing Hyg concentration, the proliferation ploidy of Pinus koraiensis embryonic callus showed a declining trend after 15 days (Figure 2). The results revealed that 4 mg·L -1 of Hyg reduced the proliferation ploidy of embryonic callus by approximately 51.72%. Furthermore, Hyg concentrations equal to or exceeding 20 mg·L -1 led to a notable reduction in embryonic callus proliferation, resulting in negative growth. Specifically, at Hyg concentrations of 20, 40, and 60 mg·L -1 in the medium, the proliferation rates of embryonic callus were reduced by 9.59, 47.94, and 56.35%, respectively. Consequently, 10 mg·L -1 of Hyg was considered the optimal screening antibiotic concentration for Pinus koraiensis embryonic callus (p < 0.01). Effects of Infiltration Solution Concentrations The efficiency of Agrobacterium -mediated transformation in Pinus koraiensis embryonic callus demonstrated a trend of increasing and then decreasing with the concentration of the infiltration solution. Upon varying the concentration of the solution, the highest conversion efficiency, reaching 93.42 ± 2.13%, was observed at an absorbance of 0.6 at OD 600 . Following closely, the next highest conversion efficiency was 89.67 ± 5.03% at an absorbance of 0.4 for the infiltration solution. Conversely, the transformation efficiencies were similar at 0.2 and 0.8, measuring 39.64 ± 4.42% and 42.63 ± 5.33%, respectively. These findings underscore the significant impact of infiltration solution concentration on the transformation efficiency of Pinus koraiensis embryonic callus (p < 0.001). Effects of Co-culture Cycle In a specified range, the efficiency of conversion increased with a prolonged co-culture duration. Five gradients of co-culture periods ranging from zero to four days were devised to determine the optimal co-culture cycles. The results indicated that absence of resistant tissues on tissue blocks screened immediately following decolonization without co-culture post-infiltration. The highest transformation efficiency, at 82.61%, was attained with a two-day co-culture. Extending the co-culture beyond three days resulted in a larger Agrobacterium presence, impacting the proliferation and growth of embryonic callus. Additionally, it became challenging to completely suppress Agrobacterium growth during the later stages of decolonization, even with the application of 500 mg·L -1 of Cef. Subsequent repeated sterilization further decreased transformation efficiency. Effects of Antibiotic Species Screening The selection of antibiotics for screening significantly impacts the efficiency of genetic transformation. Based on the results of the antibiotic susceptibility test, it was noted that Hyg demonstrated a stronger inhibitory effect on the embryonic callus of Pinus koraiensis compared to Kan. This study aimed to evaluate the screening efficacy of these antibiotics during genetic transformation. The findings demonstrated that the false positive rate of resistant tissues screened on medium containing 100 mg·L -1 Kan was approximately 71.05%, whereas the false positive rate with 10 mg·L -1 Hyg screening was only 13.89%. Furthermore, Hyg displayed a more rapid inhibitory effect on embryonic callus compared to Kan. Screening on medium containing 10 mg·L -1 Hyg for ten days led to significant browning of untransformed tissues, whereas the changes observed with medium containing 100 mg·L -1 Kan were not significant. In summary, Hyg proved more effective and efficient than Kan and was better suited for screening transformed tissues of Pinus koraiensis . GUS Staining of Resistant Tissues Resistant Pinus koraiensis embryonic callus, cultured on a screening medium containing 10 mg·L -1 Hyg for 63 days, underwent the β-glucosidase gene (GUS) histochemical staining, with uninfiltrated tissues serving as negative controls. GUS-positive embryonic callus appeared blue, indicating the successful transfer of the GUS-containing reporter gene into Pinus koraiensis embryonic callus (Figure 3). Along with the 36 embryonic callus samples examined, 32 displayed blue staining, resulting in a staining rate of 88.89%. PCR of Resistant Tissues 14 out of the 32 Hyg-resistant embryonic callus samples from Pinus koraiensis were randomly selected, and total DNA was extracted to serve as a template for PCR, aiming to confirm the successful transfer of the GUS reporter gene. The primers were designed based on the 35S promoter sequence, while uninfiltrated tissues and Agrobacterium tumefaciens served were used as negative and positive controls, respectively. The results revealed that all 14 resistant embryonic callus samples displayed a target band with a fragment length of 256 bp (Figure 4a). To evaluate the stability of the genetic transformation in this study, the LobHLH34 gene, previously cloned in the authors’ laboratory with a size of 696 bp, was transformed using the aforementioned procedure. The resulting material was subjected to GUS staining and PCR analysis. The outcomes confirmed the successful transfer of the LobHLH34 gene into Pinus koraiensis embryonic callus, thereby validating the stability of the established genetic transformation system for Pinus koraiensis (Figure 4b). Effects of Gellan Gum on Somatic Embryo Maturation The influence of gellan gum concentration on somatic embryo maturation is presented in Figure 5a. Post-screening and testing, the Pinus koraiensis resistant embryogenic callus was placed on a gellan gum medium containing 12 g·L -1 . The Pinus koraiensis embryogenic callus exhibited significant drying, with the majority of tissue turning dark brown, resulting in a somatic embryogenesis rate of 4.33 g -1 . On gellan gum medium containing 10 g·L -1 , the Pinus koraiensis embryogenic callus appeared dry and granular, with approximately half turning dark brown. Somatic embryos were observed as white globular or elongated structures, with multiple somatic embryos appearing connected, leading to a somatic embryogenesis rate of 17.33 g -1 . On gellan gum medium containing 8 g·L -1 , the embryogenic callus of Pinus koraiensis exhibited a dry and powdery texture, with predominantly white long strips distributed uniformly. The somatic embryogenesis rate reached 21.67 g -1 (Table 2). Without altering the concentration of the gellan gum medium (8, 10, and 12 g·L -1 ) (Figure 5b), the basal medium was switched from DCR to mLV (Supplementary File 2). The occurrence of somatic embryos was lower than observed in DCR culture, with rates of 11.67, 11, and 1.33 g·L -1 , respectively. However, the number of mature malformed Pinus koraiensis somatic embryos was higher than that of normal somatic embryos in both DCR and mLV. Table 2. Somatic embryogenesis at different concentrations of gellan gum. Gellan gum concentration （g/L） Amount of somatic embryogenesis （g·L -1 ） DCR medium mLV medium 8 21.67±4.41 a 11.67±4.16 a 10 17.33±6.03 a 11.00±4.35 a 12 4.33±1.53 b 1.33±1.53 b Discussion Agrobacterium -mediated genetic transformation entails integrating target genes into a plant's genome by infiltrating the plant with Agrobacterium , facilitating their expression within the plant genome. This technique has been widely utilized due to its notable transformation efficiency and the well-established mechanisms and technologies associated with it 19 . Key factors influencing efficiency in Agrobacterium -mediated transformation include bacterial solution concentration, infiltration duration, co-culture duration, antibiotic screening, and the choice of explants. Fine-tuning these factors and identifying optimal thresholds can amplify transformation efficiency, yet excessive adjustments may potentially diminish efficiency. Various plant tissues or organs, such as roots, stems, leaves, somatic embryos, mature embryos, and immature embryos, can function as recipients for genetic transformation 20 21 . In studies on conifer genetic transformation, mature and immature zygotic embryos are commonly utilized 22 . For example, Pinus massoniana 23 and slash pine 24 have been genetically transformed using mature zygotic embryos. Gao 9 employed immature Pinus koraiensis zygotic embryos to induce embryonic callus tissue, establishing a regeneration system via somatic embryogenesis. In· the current study, the Pinus koraiensis embryonic callus was selected for genetic transformation, achieving a high transformation rate of 88.89%, with the resistant embryonic callus cultured into resistant somatic embryos. During the transformation process, both infiltration duration and concentration are critical factors for successful outcomes. Insufficient concentration of the bacterial solution leads to inadequate adherence of Agrobacterium to the plant material, resulting in low transformation efficiency. Conversely, excessively high concentrations result in the over-proliferation of Agrobacterium within the plant material during later co-culture stages, posing challenges in subsequent decolonization and potentially risking plant senescence. Infiltration duration also plays a role in transformation efficiency. Too short a duration does not permit sufficient attachment of Agrobacterium to the plant material, while excessively long infiltration periods lead to an unnecessary buildup of Agrobacterium 21 . In conifer genetic transformation systems, the bacterial solution concentrations during infiltration typically range from an OD 600 absorbance of 0.3 to 0.8, with infiltration times ranging from ten to 40 minutes. For example, some Larix species have achieved the highest transformation rate with a bacterial solution concentration of approximately OD 600 =0.6 and an infiltration time of 20 min25. Similarly, optimal results in the genetic transformation of Pinus bungeana were observed with an OD 600 =0.4 and an infiltration time of 30 min 26. Transformation of Pinus taeda exhibited a transient expression rate of around 70% with a bacteriophage concentration OD 600 =0.8 and infiltration times ranging from 15 to 30 min 27 . In this study, the most favorable outcomes were attained by using an OD 600 absorbance of 0.5 for infiltrating Pinus koraiensis embryonic callus for 20 min. The co-culture phase signifies the stage at which T-DNA integration is conveyed into the plant genome. Optimal co-culture conditions for conifers have been determined to be two to four days in dark culture at 25°C. Straying from this timeframe, either too short or too long, can diminish conversion efficiency 28 . For example, in the genetic transformation of the embryonic callus of hybrid fir 29 and white spruce 30 , the co-culture period was fixed at two days. For mature zygotic embryos of Pinus massoniana 23 , incubation spanned a total of three days. The results of this study indicated that a two-day co-culture duration yielded superior efficiency in tissue transformation compared to one, three, or four days. Prolonged co-culture beyond three days led to repetitive decolonization of tissues due to elevated bacterial concentrations, ultimately leading to tissue death. The selection of antibiotic type and its concentration are crucial factors that influence the transformation process. Sensitivity tests conducted on other plant species have revealed that employing multiple screenings enhances the transformation rate while reducing the false positive rate. In conifer genetic transformation, two primary antibiotics, Kan and Hyg, are predominantly utilized for screening. Yaupon 31 achieved resistance in tissues by conducting Kan screening at concentrations ranging from 50 to 75 mg·L -1 . A positive rate of 66.2% in obtaining resistant embryogenic callus was achieved in hybrid larch under three consecutive screens with 20 mg·L -1 of Kan 32 . The false positive rate of resistance obtained via Hyg screening in Larix olgensis was only 11.66% 33 . In this study, sensitivity experiments were carried out on the Pinus koraiensis embryonic callus, demonstrating its insensitivity to Kan. At a Kan concentration of 100 mg·L -1 , significant differences in the proliferation rate of Pinus koraiensis embryonic callus were not detected. Conversely, when screening with Hyg as the antibiotic, the proliferation rate of Pinus koraiensis embryonic callus significantly decreased. At a concentration of 4 mg·L -1 , the proliferation rate was 68.42%, and the embryonic callus barely proliferated at concentrations surpassing 20 mg·L -1 , with the group tissues dying at 40 mg·L -1 . Furthermore, the number of screenings was hypothesized to affect the detected positive conversion rate. Due to the sensitivity of embryonic callus, the initial screening yielded fewer resistant tissues and a higher false positive rate. Subsequent screenings, particularly the second and third rounds, effectively decreased the false positive rate and enhanced the conversion rate. Consequently, 36 resistants embryonic calli were obtained in this study using Hyg as the antibiotic, subjected to three consecutive screenings lasting 21 days each, resulting in a significantly low false positive rate of 11.11%. Conclusions In this study, a highly efficient genetic transformation system for Pinus koraiensis embryonic callus was successfully established and optimized, marking a significant breakthrough in this field. Agrobacterium strain GV3101 was utilized in combination with the binary vector VB191103-1905rcy, resulting in the acquisition of 36 resistant embryogenic calli under specific conditions: an OD 600 absorbance value of 0.6 for the infiltrating solution, a two-day co-culture duration, and 10 mg·L -1 Hyg as the screening antibiotic. The transformation rate reached an impressive 88.89%, as validated by GUS and PCR analyses. This innovative study not only introduced a new method for investigating the gene function of Pinus koraiensis but also established a crucial foundation for developing a regenerative genetic transformation system for Pinus koraiensis plants. Methods Plant material Ling 34 provided Pinus koraiensis embryonic material, which underwent induction and was then inoculated in a DCR medium (Supplementary File 1). This medium consisted of 0.5 mg·L -1 2,4-dichlorophenoxyacetic acid (2,4-D), 0.1 mg·L -1 6-benzyladenine (6-BA), 30 g·L -1 sucrose, 4 g·L -1 gellan gum, 0.1 mg·L -1 inositol, 0.5 mg·L -1 glutamine (Gln), 0.5 mg·L -1 casein hydrolyzed (CH), with a pH of 5.9. Cultivation occurred in darkness at 25°C, with reproductive successions carried out every two days. Agrobacterium strains The Agrobacterium strain (GV3101) and the plasmid (pBI121) used in this study were maintained in the laboratory. The vector strain utilized was VB191103-1905rcy, with the plasmid, it carried containing the β-glucosidase gene (GUS), sourced from the State Key Laboratory of Forest Genetic Breeding, Northeast Forestry University, China. Antibiotic sensitivity test of embryonic callus Following a ten-day incubation in the DCR proliferation medium, about 0.2 g of embryonic callus was transferred to the DCR medium supplemented with 0.5 mg·L -1 2,4-D, 0.1 mg·L -1 6-BA, and 30 g·L -1 sucrose. The medium also contained additional components such as 4 g·L -1 gellan gum, 0.1 mg·L -1 inositol, 0.5 mg·L -1 Gln, 0.5 mg·L -1 casein hydrolyzed, along with cefotaxime (Cef), kanamycin (Kan), or hygromycin (Hyg), at pH 5.9. Screening was conducted at 25±1°C for 15 days under dark conditions. The fresh mass of the embryonic callus was measured, and this process was repeated three times. Gradient concentrations were employed for Kan (0, 10, 20, 30, and 40 mg·L -1 ), Hyg (0, 4, 8, 10, 20, 40, and 60 mg·L -1 ), and Cef (0, 100, 200, 300, and 400 mg·L -1 ). Agrobacterium strain culture A small volume of bacterial solution was drawn up into an inoculation loop and streaked onto solid yeast extract mannitol broth medium (YEB) supplemented with 50 mg·L -1 Kan, Hyg, and Rifampicin (Rif), then cultured for two days at 28℃. Following this, single colonies were selected and transferred into 20 mL of liquid YEB medium with 20 mg·L -1 Kan and Rif, shaken at 200 r·min -1 , and cultured at 28℃ for 16-18 hours in darkness until reaching an optical density (OD 600 ) of 0.8 to 1.0. Subsequently, 1 mL of the bacterial culture was inoculated into a liquid YEB medium containing 50 mg·L -1 Kan and Rif for five to eight hours, allowing the Agrobacterium to attain the logarithmic growth phase with an OD 600 of 0.6 to 0.8. Genetic transformation The fresh bacterial solution was transferred into a sterile centrifuge tube and centrifuged (Therom, China) at 4℃ and 8,000 rpm for ten minutes to collect the bacterial pellet for the infiltration solution. Fresh embryonic callus was carefully chosen and submerged in the infiltration solution, which contained 100 µM·L -1 acetosyringone (AS). The concentration of the infiltration solution was adjusted to absorbance values of 0.4, 0.6, or 1.0 at OD 600 . After a 20-min infiltration period, the solution was poured off, and the tissue surface was gently blotted with sterile filter paper before being transferred to the co-culture medium (DCR+250 mg·L -1 Cef) for two, three, or four days at 25±1 ℃ in the dark. Following co-culture, the tissues were rinsed twice with sterile water for two minutes each, followed by two washes with a suspension containing 500 mg·L -1 Cef for 3 min each. Any excess water on the surface of the cleaned callus was carefully blotted dry with sterile filter paper. The embryonic callus was spread out into sheets on a recovery medium at 25±1°C and cultured in the dark for seven, 14, or 21 days. Following this, the resistant tissues underwent screening using a specialized screening medium. The number of resistant tissues obtained was documented over three successive screenings, each lasting 21 days. These resistant tissues were then subjected to GUS histochemical staining and PCR analysis at the molecular level. Once appropriately confirmed as resistant, the embryonic callus underwent succession culture and somatic embryo maturation (Figure 6). GUS histochemical assay The resistant tissues were carefully selected and immersed in an appropriate volume of GUS staining solution, formulated with 100 mM sodium phosphate buffer (pH=7.0), 0.5 mg·mL -1 X-Gluc (5-bromo-4-chloro-3-indolyl-β-D-glucuronide), 1% Triton X-100, 1% DMSO, and 10 mM EDTA. The staining solution was infiltrated using a vacuum pump until no bubbles were visible. The embryogenic callus underwent staining in a water bath at 37℃ for one week. Non-transformed tissues were used as the negative control, and the color development status of the embryonic callus was observed and documented. Among these, tissues displaying a blue-green color were identified as transgenic tissues, whereas colorless transparent or slightly yellow tissues were considered false-positive tissues. PCR analysis Using Pinus koraiensis resistant callus DNA as a template, genomic DNA was extracted from 14 randomly selected resistant cell lines employing the CTAB method. PCR molecular assays were carried out with upstream and downstream primers for the target genes. The upstream primer sequence was 5'-CAAAGCAAGTGGATTGATGTGAT-3', and the downstream primer sequence was 5'-AGAGAGAAAAGGGTCCTAACCAAGA-3'. The reaction mixture consisted of 10µL of Green Taq MIX, 1µL each of upstream and downstream primers, 1µL of DNA, and 7µL of ddH 2 O. The reaction conditions included denaturation at 94℃ for 3 min, followed by denaturation at 94℃ for 30 sec, annealing at 60℃ for 15 sec, extension at 72℃ for 15 sec, repeated for 35 cycles. The final extension was at 72℃ for 10 min. The PCR samples were then separated using a 1.0% (w/v) agarose gel electrophoresis with wild-type Pinus koraiensis embryonic callus as the negative control and Agrobacterium solution as the positive control. The electrophoresis results were observed and captured using a gel imager (Tanon 2500R, China). The resistant embryogenic calli were identified based on the presence of correct destination bands. Somatic embryo maturation The approach for somatic embryo maturation was adapted from Peng 35 with some changes. Three grams of various transgenic Pinus koraiensis embryonic callus samples were placed into 20 mL of liquid medium (DCR+30 g·L -1 sucrose) devoid of any hormones and incubated for seven days. Subsequently, the mixture was poured off and excess liquid was absorbed using sterile filter paper. The embryonic callus was then dried on an ultra-clean bench for ten minutes. The transgenic Pinus koraiensis embryogenic callus obtained through the aforementioned methods was transferred to a DCR maturation medium, comprising varying concentrations of gellan gum (8, 10, and 12 g·L -1 ), 20 mg·L -1 ABA, 0.1 g·L -1 inositol, 0.5 g·L -1 Gln, 0.5 g·L -1 CH, 30 g·L -1 maltose, with pH adjusted to 5.9. All somatic embryogenesis events were tallied after a 10-week period. Statistical analysis SPSS statistical analysis software was used for variance analysis, setting the significance level at p < 0.05. The tables and figures were generated using Microsoft Excel 2010. Declarations Data Availability All data in this study are available in the manuscript or the Supplementary materials. Ethical declarations The plant collection and use was in accordance with all the relevant guidelines. Funding This work was supported by Scientific and Technological Innovation 2030 -Major Project of Agricultural Biological Breeding (No.2023ZD040580204). The National Key Research and Development Program of China (No.2023YFD2200605). Author contributions statement S.L. conceived and designed the experiments; H.Z. provided the financial support; L.Y. and H.S. provided the Pinus koraiensis embryonic callus; H.H. and H.D. performed the experiments, analyzed the data, prepared the figures and tables, and they contributed equally to this work; Y.W and W.Z. reviewed drafts of the paper. All authors have read and agreed to the published version of the manuscript. Competing interests The authors declare no competing interests References Zhang Z, Zhang HG, Mo C, Zhang L. Transcriptome Sequencing Analysis and Development of EST·SSR Markers for Pinus koraiensis . Scientia Silvae Sinicae . 51:114-120 (2015) Huang YY, Zhu XQ, Zhu Y, Wang ZY. Pinus koraiensis polyphenols: structural identification, in vitro antioxidant activity, immune function and inhibition of cancer cell proliferation. Food Funct . 12(9):4176-4198, Doi:10.1039/d0fo03347b (2021). Pullman, GS, Bucalo K. Pine somatic embryogenesis using zygotic embryos as explants. Methods Mol. Biol. 710:267-91, Doi:10.1007/978-1-61737-988-8-9 (2011). Gao F, Peng CX, Wang H, Tretyakova IN, Nosov AM, Shen HL, Yang L. Key Techniques for Somatic Embryogenesis and Plant Regeneration of Pinus koraiensis . Forests. 111:9, Doi:10.3390/f11090912 (2020). Gao F, Peng CX, Zhang Y, Wang H, Shen HL, Yang L. Glutathione Plays a Positive Role in the Proliferation of Pinus koraiensis Embryogenic Cells. Int. J. Mol. Sci. 23(23):14679, Doi:10.3390/ijms232314679 (2022). Peng CX, Gao F, Wang H, Tretyakova IN, Nosov AM, Shen HL, Yang L. Morphological and Physiological Indicators for Screening Cell Lines with High Potential for Somatic Embryo Maturation at an Early Stage of Somatic Embryogenesis in Pinus Koraiensis . Plants Basel . 11(14):1867. doi:10.3390/plants11141867 (2022). Peng CX, Gao F, Wang H, Shen HL, Yang L. Optimization of maturation process for somatic embryo production and cryopreservation of embryogenic tissue in Pinus koraiensis . Plant Cell Tissue and Organ Culture . 1144:1, Doi:10.1007/s11240-020-01918-y (2020). Peng CX, Gao F, Tretyakova IN, Nosov AM, Shen HL, Yang L. Transcriptomic and Metabolomic Analysis of Korean pine Cell Lines with Different Somatic Embryogenic Potential. Int. J. Mol. Sci . 23(21):13301, Doi:10.3390/ijms232113301 (2022). Gao F, Shi YJ, Wang RR, Tretyakova IN, Nosov AM, Shen HL, Yang L. Exogenous Glutathione Promotes the Proliferation of Pinus koraiensis Embryonic Cells and the Synthesis of Glutathione and Ascorbic Acid. Plants . 11:2586-2586, Doi:10.1007/s11240-022-02348-8 (2022). Castander Olarieta A, Moncalean P, Montalban IA. Somatic Embryogenesis in Pines . Methods Mol. Biol. 25:41-56, Doi:10.1007/978-1-0716-2485-2_4 (2022). Ramkumar TR, Lenka SK, Arya SS, Bansal KC. A Short History and Perspectives on Plant Genetic Transformation. Methods Mol. Biol. 2124:39-68, Doi:10.1007/978-1-0716-0356-7_3 (2020). Huang Y, Diner AM, Karnosky DF. Agrobacterium rhizogenes-mediated genetic transformation and regeneration of a conifer: Larix decidua . In Vitro Cellular & Developmental Biology - Plant . 27:201-7 (1991). Maruyama TE, Hosoi Y. Progress in Somatic Embryogenesis of Japanese Pines . Front Plant Sci . 10:31, Doi:10.3389/fpls.2019.00031 (2019). Dang SF, Zhang LF, Han SY, Qi LW. Agrobacterium -Mediated Genetic Transformation of Larix kaempferi ( Lamb. ) Carr . Embryogenic Cell Suspension Cultures and Expression Analysis of Exogenous Genes. Forests . 13(9):1436-1436, Doi:10.3390/f13091436 (2022). Tang W. (2001). Agrobacterium -mediated transformation and assessment of factors influencing transgene expression in loblolly pine (Pinus taeda L.). Cell Research . 11(3):237-43, Doi:10.1038/sj.cr.7290092 (2001). Cui Y, Zhao J, Gao Y, Zhao R, Zhang J, Kong L. Efficient Multi-Sites Genome Editing and Plant Regeneration via Somatic Embryogenesis in Picea glauca . Front Plant Sci . 12:751-891, Doi:10.3389/fpls.2021.751891 (2021). Hazubska-Przybyl T, Wawrzyniak MK, Kijowska-Oberc J, Staszak AM, RRatajczak E. Somatic Embryogenesis of Norway Spruce and Scots Pine : Possibility of Application in Modern Forestry. Forests. 13:2, Doi:10.3390/f13020155 (2022). Liang Y, Xu X, Shen HL, Gao ML, Zhao Y, Bai X. Morphological and endogenous phytohormone changes during long-term embryogenic cultures in Korean pine . Plant Cell Tissue and Organ Culture . 151:253-264, Doi:10.1007/s11240-022-02348-8 (2022). Peng CX, Gao F, Wang H, Shen HL, Yang L. Physiological and Biochemical Traits in Korean pine Somatic Embryogenesis. Forests . 11(5): 577-577, Doi10.3390/f1105057 (2020). Wang B, Zhang Z, Yin Z, Feng C, Wang Q. Novel and potential application of cryopreservation to plant genetic transformation. Biotechnol. Adv . 30(3):604-12, Doi:10.1016/j. biotechadv.2011.10.008 (2011). Yao R, Wang Y. An advanced protocol for the establishment of plantlets originating from somatic embryos in Pinus massoniana . 3 Biotech . 10(9):394, Doi:10.1007/s13205-020-02385-0 (2020). Zhang Y. Establishment of genetic transformation receptor systems and Agrobacterium -mediated genetic transformation in three coniferous species. Dissertation for the Degree. Shanghai: Graduate School of Chinese Academy of Sciences (2006). Maleki SS, Mohammadi K, Ji KS. Study on factors influencing transformation efficiency in Pinus massoniana using Agrobacterium tumefaciens. Plant Cell Tissue and Organ Culture. 133(3):437-445, Doi:10.1007/s11240-018-1388-7 (2018). Tang. W, Xiao B, Fei YJ. Slash pine genetic transformation through embryo cocultivation with a tumefaciens and transgenic plant regeneratio. I n Vitro Cellular & Developmental Biology. Plant . 50(2): 199-209 (2014). Lin XF, Zhang WB, Takechi K, Takio S, Ono, Takano H. Stable genetic transformation of Larix gmelinii L. by particle bombardment of zygotic embryos. Plant Cell Rep . 24:418-425, Doi:10.1007/s00299-005-0955-7 (2005). Zhang L, Qi LW, Han SY. Differentially expressed genes during Larix somatic embryo maturation and the expression profile of partial genes. Yi chuan Hereditas . 31:540-5 (2009). Tang W, Newton RJ. Genetic transformation of conifers and its application in forest biotechnology. Plant cell Reports . 22(1):1-15, Doi: 10.1007/s00299-003-0670-1 (2003). Ramkumar TR, Lenka SK, Arya SS, Bansal KC. A Short History and Perspectives on Plant Genetic Transformation. Methods Mol Biol . 24:39-68, Doi:10.1007/978-1-0716-0356-7_3 (2020). Salaj T, Moravcikova J, Vookova B, Salaj J. Agrobacterium -mediated transformation of embryogenic tissues of hybrid firs ( Abies spp .) and regeneration of transgenic emblings. Biotechnology letters . 331:647-652, Doi:10.1007/s10529-009-9923-6 (2009). Le VQ, Belles-Isles J, Dusabenyagasani M, Trembiay FM. An improved procedure for production of white spruce ( Picea glauca ) transgenic plants using Agrobacterium tumefaciens. Journal of experimental botany . 52:2089-2095, Doi:10.1093/jexbot/52.364.2089 (2001). Liu S, Ma J, Liu H, Guo Y, Li W, Niu S. An efficient system for Agrobacterium -mediated transient transformation in Pinus tabuliformis. Plant Methods. 16:52, Doi:10.1186/s13007-020-00594-5 (2020). Gleeson D, Lelu-Walter M, Parkinson M. Overproduction of proline in transgenic hybrid larch (Larix x leptoeuropaea (Dengler)) cultures renders them tolerant to cold, salt and frost. Molecular Breeding, 15:21-29, Doi:10.1007/s11032-004-1363-3 (2005). Song Y. et al. Stable and Efficient Agrobacterium -Mediated Genetic Transformation of Larch Using Embryogenic Callus. Frontiers in Plant Science. 11: 84492-584492, Doi:10.3389/fpls.2020.584492 (2020). Gao F, Peng CX, Wang H, Shen HL, Yang L. Selection of culture conditions for callus induction and proliferation by somatic embryogenesis of Pinus koraiensi . Journal of Forestry Researc h. 32(02):483-491, Doi10.1007/s11676-020-01147-1 (2021). Peng CX, Gao F, Wang H, Tretyakova IN, Nosov AM, Shen H.L, Yang L. Suspension Culture and Somatic Embryogenesis of Korean pine . Phyton. Int. J. Exp. Bot. 91:223-238, Doi:10.32604/phyton.2022.015523 (2022). Additional Declarations No competing interests reported. Supplementary Files Supplementary.docx Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4173927","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":290157318,"identity":"a40ff8cb-99c4-4114-a38b-43f23e23a45a","order_by":0,"name":"Hui Hou","email":"","orcid":"","institution":"State Key Laboratory of Tree Genetics and Breeding, Northeast Forestry University","correspondingAuthor":false,"prefix":"","firstName":"Hui","middleName":"","lastName":"Hou","suffix":""},{"id":290157321,"identity":"6046228c-945c-4d39-8892-dfc935e0f839","order_by":1,"name":"Hao Dong","email":"","orcid":"","institution":"State Key Laboratory of Tree Genetics and Breeding, Northeast Forestry University","correspondingAuthor":false,"prefix":"","firstName":"Hao","middleName":"","lastName":"Dong","suffix":""},{"id":290157325,"identity":"df7c4cdb-a66c-40c6-8cf7-8a3af22e785a","order_by":2,"name":"Yanan Wu","email":"","orcid":"","institution":"State Key Laboratory of Tree Genetics and Breeding, Northeast Forestry University","correspondingAuthor":false,"prefix":"","firstName":"Yanan","middleName":"","lastName":"Wu","suffix":""},{"id":290157326,"identity":"0f17821c-115b-4308-a2f1-74a6d071bfcf","order_by":3,"name":"Wenna Zhao","email":"","orcid":"","institution":"State Key Laboratory of Tree Genetics and Breeding, Northeast Forestry University","correspondingAuthor":false,"prefix":"","firstName":"Wenna","middleName":"","lastName":"Zhao","suffix":""},{"id":290157330,"identity":"c5d7dcf8-8fce-4553-918f-8a2f78aba635","order_by":4,"name":"Hailong Shen","email":"","orcid":"","institution":"School of Forestry, Northeast Forestry University","correspondingAuthor":false,"prefix":"","firstName":"Hailong","middleName":"","lastName":"Shen","suffix":""},{"id":290157332,"identity":"797409f6-e804-4227-83d2-0fddce279b02","order_by":5,"name":"Ling Yang","email":"","orcid":"","institution":"School of Forestry, Northeast Forestry University","correspondingAuthor":false,"prefix":"","firstName":"Ling","middleName":"","lastName":"Yang","suffix":""},{"id":290157333,"identity":"57240fb5-51f2-4825-b960-3ef9f4d6b205","order_by":6,"name":"Hanguo Zhang","email":"","orcid":"","institution":"State Key Laboratory of Tree Genetics and Breeding, Northeast Forestry University","correspondingAuthor":false,"prefix":"","firstName":"Hanguo","middleName":"","lastName":"Zhang","suffix":""},{"id":290157334,"identity":"07e7bf8b-5df1-421b-8887-cb4a341ecf81","order_by":7,"name":"Shujuan Li","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA50lEQVRIiWNgGAWjYBACPmYGNgYGHiCLvYGBoQIkdICAFja4Fh6g0jNEaQEjEJBIIFYLO/uzxzwyh+XNJd8efHCwjUGO70YC4+cCvA7jMTfm4TlsuHN2XrIBUIux5I0EZukZ+LWwSQO1MG64nWMm/bGNIXHDjQSgIF4t7M9AWuw33Dxj/gNoSz0RWhjMQFqAhvOYMQC1JBgQ1sJjJjmHJz15w5kcY4kD5yQMZ5552CyNTws///FnEm97rG03HD9j+OFAmY083/Hkg5/xaQEDxh44UwLEbSCkAQh+EKFmFIyCUTAKRi4AAM85RW/kk555AAAAAElFTkSuQmCC","orcid":"","institution":"State Key Laboratory of Tree Genetics and Breeding, Northeast Forestry University","correspondingAuthor":true,"prefix":"","firstName":"Shujuan","middleName":"","lastName":"Li","suffix":""}],"badges":[],"createdAt":"2024-03-27 06:42:12","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4173927/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4173927/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":54579930,"identity":"2a671b45-734e-4589-a9a6-7ebc1ce0c6ac","added_by":"auto","created_at":"2024-04-12 14:24:15","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":278716,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of Kan on embryonic callus. (a-f) Proliferation at 0, 4, 10, 20, 40, and 60 mg/L for 15 days of culture. (g) The proliferation rate of the callus with different Kan concentrations, bar=1 cm.\u003c/p\u003e","description":"","filename":"image1.png","url":"https://assets-eu.researchsquare.com/files/rs-4173927/v1/d3c251516437181b64514424.png"},{"id":54580780,"identity":"34f2be0f-33da-47c6-b669-5c99845cb19e","added_by":"auto","created_at":"2024-04-12 14:32:14","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":227351,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of Hyg on embryonic callus. (a-f) Proliferation at 0, 4, 10, 20, 40, and 60 mg/L for 15 days of culture. (g) The proliferation rate of callus with different Hyg concentrations, bar=1 cm.\u003c/p\u003e","description":"","filename":"image2.png","url":"https://assets-eu.researchsquare.com/files/rs-4173927/v1/be8972a717ace4dd5edb45b8.png"},{"id":54579929,"identity":"c4ed581c-13fe-4f80-a5b6-9b23b4512449","added_by":"auto","created_at":"2024-04-12 14:24:14","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":559256,"visible":true,"origin":"","legend":"\u003cp\u003eGUS staining of resistant embryonic callus. (a,1-3) GUS staining of wild-type embryonic callus (a,4-6) GUS staining of resistant callus(a,7-9) GUS staining of \u003cem\u003eLobHLH\u003c/em\u003egene (b) GUS staining of wild-type embryonic callus (c) GUS staining of resistant callus (d) GUS staining of \u003cem\u003eLobHLH\u003c/em\u003e gene.\u003c/p\u003e","description":"","filename":"image3.png","url":"https://assets-eu.researchsquare.com/files/rs-4173927/v1/756097154df01a2e36955fe4.png"},{"id":54579925,"identity":"ec3f533c-5d99-46ce-b732-365e9c9f53ec","added_by":"auto","created_at":"2024-04-12 14:24:13","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":147229,"visible":true,"origin":"","legend":"\u003cp\u003ePCR Assay. (a) PCR assay of resistant callus with GV3101 vector (b) PCR assay of \u003cem\u003eLobHLH\u003c/em\u003egene.\u003c/p\u003e","description":"","filename":"image4.png","url":"https://assets-eu.researchsquare.com/files/rs-4173927/v1/ca2aec6e42eca182a3997040.png"},{"id":54580782,"identity":"2acb1b31-a119-4e4a-a9ac-81d597a3869d","added_by":"auto","created_at":"2024-04-12 14:32:15","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":508208,"visible":true,"origin":"","legend":"\u003cp\u003eOccurrence of resistant somatic embryos. (a) somatic embryogenesis in DCR medium with Gellan gum concentrations of 8, 10 and 12 g/L (b) somatic embryogenesis in mLV medium with Gellan gum concentrations of 8, 10 and 12 g/L, bar=1 cm.\u003c/p\u003e","description":"","filename":"image5.png","url":"https://assets-eu.researchsquare.com/files/rs-4173927/v1/4a8a2384961493772a0a8be6.png"},{"id":54579926,"identity":"ab9d591f-ebc5-46ef-931f-26b77adb81cc","added_by":"auto","created_at":"2024-04-12 14:24:14","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":543991,"visible":true,"origin":"","legend":"\u003cp\u003eGenetic transformation process. (a) Successional culture (b) \u003cem\u003eAgrobacterium\u003c/em\u003e infestation (c) Co-culture (d) Recovery Culture (e) First screening culture (f) Second screening culture (g) Third screening culture (h) Obtaining resistant somatic embryos.\u003c/p\u003e","description":"","filename":"image6.png","url":"https://assets-eu.researchsquare.com/files/rs-4173927/v1/1126bdca0f5c0d40ba74a249.png"},{"id":66931593,"identity":"0a4b9792-d7fe-454f-8df9-1a5aef6b9c33","added_by":"auto","created_at":"2024-10-18 07:09:26","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3404888,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4173927/v1/3a18aa4e-601c-4854-a608-b00c29518c14.pdf"},{"id":54581358,"identity":"2bc8abd5-cb3c-4a53-a97d-e387f89ca902","added_by":"auto","created_at":"2024-04-12 14:40:14","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":18186,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementary.docx","url":"https://assets-eu.researchsquare.com/files/rs-4173927/v1/8a7fd274be21a207d8c05328.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"A method for permanent genetic transformation using embryonic callus of Pinus koraiensis","fulltext":[{"header":"Introduction","content":"\u003cp\u003eScientifically\u0026nbsp;classified as an evergreen tree within the\u0026nbsp;\u003cem\u003ePinus\u003c/em\u003e genus of the\u0026nbsp;Pinaceae family,\u0026nbsp;\u003cem\u003ePinus koraiensis\u003c/em\u003e, also known\u0026nbsp;as the sea pine,\u0026nbsp;is naturally distributed across China,\u0026nbsp;particularly in\u0026nbsp;the Great and Small Xing\u0026apos;an Mountains and the Changbai Mountains\u003csup\u003e1\u003c/sup\u003e\u003csup\u003e.\u0026nbsp;\u003c/sup\u003eIn\u0026nbsp;northeastern China, \u003cem\u003ePinus koraiensis\u003c/em\u003e holds\u0026nbsp;significant\u0026nbsp;importance\u0026nbsp;as\u0026nbsp;a\u0026nbsp;native timber species.\u0026nbsp;Its significance extends beyond timber, as its sapwood, bark, pine needles, and cones\u0026nbsp;serve\u0026nbsp;as\u0026nbsp;crucial\u0026nbsp;industrial raw materials\u003csup\u003e2\u003c/sup\u003e\u003csup\u003e.\u003c/sup\u003e\u003c/p\u003e\n\u003cp\u003eAside from\u0026nbsp;its\u003csup\u003e\u0026nbsp;\u003c/sup\u003eindustrial\u0026nbsp;utility,\u0026nbsp;\u003cem\u003ePinus koraiensis\u003c/em\u003e fruit\u0026nbsp;is renowned\u0026nbsp;for its rich nutritional content,\u0026nbsp;making\u0026nbsp;it a\u0026nbsp;cornerstone of\u0026nbsp;both the nut economy and timber forest sectors in China.\u0026nbsp;Recognized for its\u0026nbsp;ecological, economic, medicinal, and nutritional\u0026nbsp;value\u0026nbsp;\u003csup\u003e3\u003c/sup\u003e,\u0026nbsp;\u003cem\u003ePinus koraiensis\u003c/em\u003e has\u0026nbsp;been the subject of\u0026nbsp;research focusing on genetic diversity, seedling reforestation, and resource utilization.\u0026nbsp;Recent\u0026nbsp;years have\u0026nbsp;seen increased\u0026nbsp;attention towards\u0026nbsp;propagating\u0026nbsp;and\u0026nbsp;genetically enhancing\u0026nbsp;\u003cem\u003ePinus koraiensis\u003c/em\u003e due to its substantial ecological and economic contributions.\u003c/p\u003e\n\u003cp\u003eDespite facing\u0026nbsp;challenges in widespread use for large-scale silvicultural seedling production\u0026nbsp;due to its extended growth cycle and limited asexual propagation techniques, \u003cem\u003ePinus koraiensis\u003c/em\u003e shows promise in\u0026nbsp;somatic embryogenesis. This technique, known for its stable heritability, rapid reproduction, and low mutation rate,\u0026nbsp;offers potential\u0026nbsp;for both the regeneration and genetic improvement of \u003cem\u003ePinus koraiensis\u003c/em\u003e.\u0026nbsp;Researchers like\u0026nbsp;Gao\u003csup\u003e4\u003c/sup\u003e\u003csup\u003e,\u003c/sup\u003e\u003csup\u003e5\u003c/sup\u003e have successfully\u0026nbsp;generated\u0026nbsp;\u003cem\u003ePinus koraiensis\u003c/em\u003e embryonic callus from immature seed embryos,\u0026nbsp;leading to plant regeneration\u0026nbsp;through somatic embryogenesis.\u0026nbsp;Additionally, Peng\u0026nbsp;et al.\u0026nbsp;\u003csup\u003e6\u003c/sup\u003e\u003csup\u003e-\u003c/sup\u003e\u003csup\u003e9\u003c/sup\u003e\u003csup\u003e\u0026nbsp;\u003c/sup\u003eutilized \u003cem\u003ePinus koraiensis\u003c/em\u003e embryonic callus to establish an ultra-low temperature preservation system and\u0026nbsp;explored\u0026nbsp;proliferation differences using transcriptome and metabolomics sequencing technology.\u0026nbsp;These advancements refine crucial\u0026nbsp;technological aspects of \u003cem\u003ePinus koraiensis\u003c/em\u003e somatic embryogenesis\u003csup\u003e10\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eThe \u003cem\u003eAgrobacterium\u003c/em\u003e-mediated genetic transformation\u0026nbsp;method has emerged\u0026nbsp;as the most extensively\u0026nbsp;studied, technically\u0026nbsp;advanced, efficient, and widely\u0026nbsp;utilized approach\u0026nbsp;for genetic transformation in conifers\u003csup\u003e11\u003c/sup\u003e.\u0026nbsp;Notably, Huang\u0026nbsp;\u003csup\u003e12\u003c/sup\u003e pioneered the\u0026nbsp;inoculation\u0026nbsp;of sterile European larch seedlings with hairy \u003cem\u003eAgrobacterium\u003c/em\u003e, leading to the successful production of\u0026nbsp;transformed plants.\u0026nbsp;Presently, transgenic embryogenic cultures have been\u0026nbsp;effectively developed using\u0026nbsp;the \u003cem\u003eAgrobacterium\u003c/em\u003e-mediated\u0026nbsp;technique\u0026nbsp;in various tree species, including Japanese larch (\u003cem\u003eLarix kaempferi\u003c/em\u003e),\u0026nbsp;torch\u0026nbsp;pine (\u003cem\u003ePinus taeda\u003c/em\u003e),\u0026nbsp;and Norway spruce (\u003cem\u003ePicea abies\u003c/em\u003e)\u0026nbsp;\u003csup\u003e13\u003c/sup\u003e\u003csup\u003e-\u003c/sup\u003e\u003csup\u003e17\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eBy integrating the somatic embryogenesis pathway with genetic transformation technology\u0026nbsp;\u003csup\u003e18\u003c/sup\u003e, the\u0026nbsp;utilization of\u0026nbsp;somatic embryogenesis\u0026nbsp;enables\u0026nbsp;the\u0026nbsp;conversion\u0026nbsp;of resistant callus tissues identified during genetic transformation into somatic embryos. These embryos can\u0026nbsp;subsequently\u0026nbsp;germinate to\u0026nbsp;yield\u0026nbsp;fully resistant plants, offering\u0026nbsp;potentially higher\u0026nbsp;efficiency\u0026nbsp;compared to\u0026nbsp;traditional organ explant transformation and\u0026nbsp;generating\u0026nbsp;purer transformed plants.\u003c/p\u003e\n\u003cp\u003eIn\u0026nbsp;this\u0026nbsp;study, \u003cem\u003eAgrobacterium\u003c/em\u003e strain GV3101\u0026nbsp;was utilized\u0026nbsp;along with the binary vector VB191103-1905rcy,\u0026nbsp;employing\u0026nbsp;\u003cem\u003ePinus koraiensis\u003c/em\u003e embryonic callus as the primary material.\u0026nbsp;Through optimization of\u0026nbsp;bacterial solution\u0026nbsp;concentrations\u0026nbsp;and infiltration\u0026nbsp;times\u0026nbsp;during the transformation process, resistant embryonic\u0026nbsp;calli were successfully obtained. This established a straightforward and efficient \u003cem\u003eAgrobacterium\u003c/em\u003e-mediated genetic transformation system for \u003cem\u003ePinus koraiensis\u003c/em\u003e embryonic tissue is poised to\u0026nbsp;accelerate\u0026nbsp;transgenesis development and\u0026nbsp;enhance\u0026nbsp;research into genetic improvement and molecular functions of \u003cem\u003ePinus koraiensis\u003c/em\u003e.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cstrong\u003eCef Effects on Embryonic Callus\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCef did not\u0026nbsp;significantly\u0026nbsp;impact the inhibition of \u003cem\u003ePinus koraiensis\u003c/em\u003e embryonic callus\u0026nbsp;(Table 1). With increasing Cef\u0026nbsp;concentration, the proliferation rate of \u003cem\u003ePinus koraiensis\u003c/em\u003e embryonic callus\u0026nbsp;gradually decreased\u0026nbsp;after 15 days. At an\u0026nbsp;additional\u0026nbsp;concentration of 500 mg\u0026middot;L\u003csup\u003e-1\u003c/sup\u003e, the fresh mass of\u0026nbsp;the\u0026nbsp;proliferating embryonic callus\u0026nbsp;decreased\u0026nbsp;by 10.01% compared to the control. The inhibitory effects on the embryonic callus of \u003cem\u003ePinus koraiensis\u003c/em\u003e were not significant across all tested\u0026nbsp;Cef treatment concentrations (p \u0026gt; 0.05).\u003c/p\u003e\n\u003cp\u003eTable 1.\u0026nbsp;Different concentrations of Cef on the proliferation rate of embryonic callus\u0026nbsp;\u003c/p\u003e\n\u003ctable border=\"0\" cellspacing=\"0\" cellpadding=\"0\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd width=\"51.845018450184504%\" valign=\"top\"\u003e\n \u003cp\u003eCef concentration (mg\u0026middot;L\u003csup\u003e-1\u003c/sup\u003e)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"48.154981549815496%\" valign=\"top\"\u003e\n \u003cp\u003eRate of Callus proliferation (%)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"51.845018450184504%\" valign=\"top\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"48.154981549815496%\" valign=\"top\"\u003e\n \u003cp\u003e3.36\u0026plusmn;0.32 a\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"51.845018450184504%\" valign=\"top\"\u003e\n \u003cp\u003e100\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"48.154981549815496%\" valign=\"top\"\u003e\n \u003cp\u003e3.28\u0026plusmn;0.49 a\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"51.845018450184504%\" valign=\"top\"\u003e\n \u003cp\u003e200\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"48.154981549815496%\" valign=\"top\"\u003e\n \u003cp\u003e3.18\u0026plusmn;0.43 a\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"51.845018450184504%\" valign=\"top\"\u003e\n \u003cp\u003e300\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"48.154981549815496%\" valign=\"top\"\u003e\n \u003cp\u003e3.00\u0026plusmn;0.39 a\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"51.845018450184504%\" valign=\"top\"\u003e\n \u003cp\u003e400\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"48.154981549815496%\" valign=\"top\"\u003e\n \u003cp\u003e3.07\u0026plusmn;0.75 a\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"51.845018450184504%\" valign=\"top\"\u003e\n \u003cp\u003e500\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"48.154981549815496%\" valign=\"top\"\u003e\n \u003cp\u003e2.87\u0026plusmn;0.42 a\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u003cbr\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eKan Effects on Embryonic Callus\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThere were no significant differences in the proliferation rate of \u003cem\u003ePinus koraiensis\u003c/em\u003e embryonic callus with increasing Kan concentration (Figure 1). The proliferation ploidy of embryonic callus cultured for 15 days without Kan was 3.35. Upon adding Kan at concentrations of 10, 20, 40, 80, and 100 mg\u0026middot;L\u003csup\u003e-1\u003c/sup\u003e, the proliferation rates were 2.89, 2.57, 2.59, 2.52, and 2.07, respectively.\u0026nbsp;A\u0026nbsp;significant difference (p \u0026lt; 0.05) in embryonic callus\u0026nbsp;proliferation was observed only\u0026nbsp;when the Kan concentration in the medium\u0026nbsp;reached\u0026nbsp;100 mg\u0026middot;L\u003csup\u003e-1\u003c/sup\u003e. However, no significant difference in the proliferation of \u003cem\u003ePinus koraiensis\u003c/em\u003e embryonic callus was noted when Kan concentration was below 100 mg\u0026middot;L\u003csup\u003e-1\u003c/sup\u003e.\u0026nbsp;Inhibitory effects were evident only at Kan\u0026nbsp;concentrations\u0026nbsp;exceeding\u0026nbsp;100 mg\u0026middot;L\u003csup\u003e-1\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eHyg Effects on Embryonic Callus\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eHyg exerted a significant inhibitory effect on the proliferation of \u003cem\u003ePinus koraiensis\u003c/em\u003e embryonic callus. With increasing Hyg concentration, the proliferation ploidy of \u003cem\u003ePinus koraiensis\u003c/em\u003e embryonic callus showed a declining trend after 15 days (Figure 2). The results revealed that 4 mg\u0026middot;L\u003csup\u003e-1\u003c/sup\u003e of Hyg reduced the proliferation ploidy of embryonic callus by approximately 51.72%. Furthermore, Hyg concentrations equal to or exceeding 20 mg\u0026middot;L\u003csup\u003e-1\u003c/sup\u003e led to a notable reduction in embryonic callus proliferation, resulting in negative growth. Specifically, at Hyg concentrations of 20, 40, and 60 mg\u0026middot;L\u003csup\u003e-1\u003c/sup\u003e in the medium, the proliferation rates of embryonic callus were reduced by 9.59, 47.94, and 56.35%, respectively. Consequently, 10 mg\u0026middot;L\u003csup\u003e-1\u003c/sup\u003e of Hyg was considered the optimal screening antibiotic concentration for \u003cem\u003ePinus koraiensis\u003c/em\u003e embryonic callus (p \u0026lt; 0.01).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEffects of Infiltration Solution Concentrations\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe efficiency of \u003cem\u003eAgrobacterium\u003c/em\u003e-mediated transformation in \u003cem\u003ePinus koraiensis\u003c/em\u003e embryonic callus demonstrated a trend of increasing and then decreasing with the concentration of the infiltration solution. Upon varying the concentration of the solution, the highest conversion efficiency, reaching 93.42 \u0026plusmn; 2.13%, was observed at an absorbance of 0.6 at OD\u003csub\u003e600\u003c/sub\u003e. Following closely, the next highest conversion efficiency was 89.67 \u0026plusmn; 5.03% at an absorbance of 0.4 for the infiltration solution. Conversely, the transformation efficiencies were similar at 0.2 and 0.8, measuring 39.64 \u0026plusmn; 4.42% and 42.63 \u0026plusmn; 5.33%, respectively. These findings underscore the significant impact of infiltration solution concentration on the transformation efficiency of \u003cem\u003ePinus koraiensis\u003c/em\u003e embryonic callus (p \u0026lt; 0.001).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEffects of Co-culture Cycle\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIn a specified range, the efficiency of conversion increased with a prolonged co-culture duration. Five gradients of co-culture periods ranging from zero to four days were devised to determine the optimal co-culture cycles. The results indicated that absence of resistant tissues on tissue blocks screened immediately following decolonization without co-culture post-infiltration. The highest transformation efficiency, at 82.61%, was attained with a two-day co-culture. Extending the co-culture beyond three days resulted in a larger \u003cem\u003eAgrobacterium\u003c/em\u003e presence, impacting the proliferation and growth of embryonic callus. Additionally, it became challenging to completely suppress \u003cem\u003eAgrobacterium\u003c/em\u003e growth during the later stages of decolonization, even with the application of 500 mg\u0026middot;L\u003csup\u003e-1\u003c/sup\u003e of Cef. Subsequent repeated sterilization further decreased transformation efficiency.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEffects of Antibiotic Species Screening\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe selection of antibiotics for screening significantly impacts the efficiency of genetic transformation. Based on the results of the antibiotic susceptibility test, it was noted that Hyg demonstrated a stronger inhibitory effect on the embryonic callus of \u003cem\u003ePinus koraiensis\u003c/em\u003e compared to Kan. This study aimed to evaluate the screening efficacy of these antibiotics during genetic transformation. The findings demonstrated that the false positive rate of resistant tissues screened on medium containing 100 mg\u0026middot;L\u003csup\u003e-1\u003c/sup\u003e Kan was approximately 71.05%, whereas the false positive rate with 10 mg\u0026middot;L\u003csup\u003e-1\u003c/sup\u003e Hyg screening was only 13.89%.\u003c/p\u003e\n\u003cp\u003eFurthermore, Hyg\u0026nbsp;displayed\u0026nbsp;a more rapid inhibitory effect on embryonic callus compared to Kan. Screening on medium containing 10 mg\u0026middot;L\u003csup\u003e-1\u003c/sup\u003e Hyg for ten days led to significant browning of untransformed tissues, whereas the changes observed with medium containing 100 mg\u0026middot;L\u003csup\u003e-1\u003c/sup\u003e Kan were not significant. In summary, Hyg proved more effective and efficient than Kan and was better suited for screening transformed tissues of \u003cem\u003ePinus koraiensis\u003c/em\u003e.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eGUS Staining of Resistant Tissues\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eResistant \u003cem\u003ePinus koraiensis\u003c/em\u003e embryonic callus, cultured on a screening medium containing 10 mg\u0026middot;L\u003csup\u003e-1\u003c/sup\u003e Hyg for 63 days, underwent the \u0026beta;-glucosidase gene (GUS) histochemical staining, with uninfiltrated tissues serving as negative controls. GUS-positive embryonic callus appeared blue, indicating the successful transfer of the GUS-containing reporter gene into \u003cem\u003ePinus koraiensis\u003c/em\u003e embryonic callus (Figure 3). Along with the 36 embryonic callus samples examined, 32 displayed blue staining, resulting in a staining rate of 88.89%.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePCR of Resistant Tissues\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e14 out of the 32 Hyg-resistant embryonic callus samples\u0026nbsp;from \u003cem\u003ePinus koraiensis\u003c/em\u003e were randomly selected, and total DNA was extracted\u0026nbsp;to serve\u0026nbsp;as a template for PCR, aiming\u0026nbsp;to\u0026nbsp;confirm\u0026nbsp;the successful transfer of the GUS reporter gene. The primers were designed\u0026nbsp;based on\u0026nbsp;the 35S promoter sequence,\u0026nbsp;while uninfiltrated\u0026nbsp;tissues and \u003cem\u003eAgrobacterium\u003c/em\u003e \u003cem\u003etumefaciens\u003c/em\u003e served\u0026nbsp;were used as negative and positive controls,\u0026nbsp;respectively.\u0026nbsp;The results\u0026nbsp;revealed\u0026nbsp;that all 14 resistant embryonic callus samples\u0026nbsp;displayed\u0026nbsp;a target band with a fragment length of 256 bp (Figure\u0026nbsp;4a).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eTo evaluate the stability of the genetic transformation in this study, the \u003cem\u003eLobHLH34\u003c/em\u003e gene, previously cloned in the authors\u0026rsquo; laboratory with a size of 696 bp, was transformed using the aforementioned procedure. The resulting material was subjected to GUS staining and PCR analysis. The outcomes confirmed the successful transfer of the \u003cem\u003eLobHLH34\u003c/em\u003e gene into \u003cem\u003ePinus koraiensis\u003c/em\u003e embryonic callus, thereby validating the stability of the established genetic transformation system for \u003cem\u003ePinus koraiensis\u003c/em\u003e (Figure 4b).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEffects of Gellan Gum on Somatic Embryo Maturation\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe\u0026nbsp;influence\u0026nbsp;of gellan gum concentration on somatic embryo maturation is\u0026nbsp;presented\u0026nbsp;in\u0026nbsp;Figure\u0026nbsp;5a.\u0026nbsp;Post-screening and testing, the\u0026nbsp;\u003cem\u003ePinus koraiensis\u003c/em\u003e resistant embryogenic callus\u0026nbsp;was placed on\u0026nbsp;a\u0026nbsp;gellan gum medium containing 12 g\u0026middot;L\u003csup\u003e-1\u003c/sup\u003e. The \u003cem\u003ePinus koraiensis\u003c/em\u003e embryogenic callus exhibited significant drying, with\u0026nbsp;the majority of tissue\u0026nbsp;turning dark brown, resulting in\u0026nbsp;a somatic embryogenesis rate of 4.33 g\u003csup\u003e-1\u003c/sup\u003e. On gellan gum medium containing 10 g\u0026middot;L\u003csup\u003e-1\u003c/sup\u003e, the \u003cem\u003ePinus koraiensis\u003c/em\u003e embryogenic callus appeared dry and granular, with\u0026nbsp;approximately\u0026nbsp;half turning dark brown. Somatic embryos were observed as white globular or elongated structures,\u0026nbsp;with\u0026nbsp;multiple somatic embryos\u0026nbsp;appearing\u0026nbsp;connected,\u0026nbsp;leading to\u0026nbsp;a somatic embryogenesis rate of 17.33 g\u003csup\u003e-1\u003c/sup\u003e. On gellan gum medium containing 8 g\u0026middot;L\u003csup\u003e-1\u003c/sup\u003e, the embryogenic callus\u0026nbsp;of \u003cem\u003ePinus koraiensis\u003c/em\u003e exhibited\u0026nbsp;a dry and powdery texture, with\u0026nbsp;predominantly\u0026nbsp;white long strips distributed\u0026nbsp;uniformly. The somatic embryogenesis rate reached 21.67 g\u003csup\u003e-1\u003c/sup\u003e (Table 2).\u003c/p\u003e\n\u003cp\u003eWithout altering the concentration of the gellan gum medium (8, 10, and 12 g\u0026middot;L\u003csup\u003e-1\u003c/sup\u003e) (Figure\u0026nbsp;5b),\u0026nbsp;the\u0026nbsp;basal\u0026nbsp;medium was\u0026nbsp;switched\u0026nbsp;from DCR to mLV (Supplementary File 2). The occurrence of somatic embryos was lower than observed in DCR culture, with rates of 11.67, 11, and 1.33 g\u0026middot;L\u003csup\u003e-1\u003c/sup\u003e, respectively. However, the number of mature malformed \u003cem\u003ePinus koraiensis\u003c/em\u003e somatic embryos was higher than that of normal somatic embryos in both DCR and mLV.\u003c/p\u003e\n\u003cp\u003eTable 2.\u0026nbsp;Somatic embryogenesis at different concentrations of gellan gum.\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd width=\"33.273056057866185%\" rowspan=\"2\" valign=\"top\"\u003e\n \u003cp\u003eGellan gum concentration\u0026nbsp;（g/L）\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"66.72694394213381%\" colspan=\"2\" valign=\"top\"\u003e\n \u003cp\u003eAmount of somatic embryogenesis\u0026nbsp;（g\u0026middot;L\u003csup\u003e-1\u003c/sup\u003e）\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"50%\" valign=\"top\"\u003e\n \u003cp\u003eDCR medium\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"50%\" valign=\"top\"\u003e\n \u003cp\u003emLV medium\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"33.333333333333336%\" valign=\"top\"\u003e\n \u003cp\u003e8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"33.333333333333336%\" valign=\"top\"\u003e\n \u003cp\u003e21.67\u0026plusmn;4.41 a\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"33.333333333333336%\" valign=\"top\"\u003e\n \u003cp\u003e11.67\u0026plusmn;4.16 a\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"33.333333333333336%\" valign=\"top\"\u003e\n \u003cp\u003e10\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"33.333333333333336%\" valign=\"top\"\u003e\n \u003cp\u003e17.33\u0026plusmn;6.03 a\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"33.333333333333336%\" valign=\"top\"\u003e\n \u003cp\u003e11.00\u0026plusmn;4.35 a\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"33.333333333333336%\" valign=\"top\"\u003e\n \u003cp\u003e12\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"33.333333333333336%\" valign=\"top\"\u003e\n \u003cp\u003e4.33\u0026plusmn;1.53 b\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"33.333333333333336%\" valign=\"top\"\u003e\n \u003cp\u003e1.33\u0026plusmn;1.53 b\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e"},{"header":"Discussion","content":"\u003cp\u003e\u003cem\u003eAgrobacterium\u003c/em\u003e-mediated genetic transformation\u0026nbsp;entails\u0026nbsp;integrating target genes into a plant\u0026apos;s genome by\u0026nbsp;infiltrating\u0026nbsp;the plant with \u003cem\u003eAgrobacterium\u003c/em\u003e,\u0026nbsp;facilitating\u0026nbsp;their expression\u0026nbsp;within\u0026nbsp;the plant genome. This\u0026nbsp;technique has been\u0026nbsp;widely\u0026nbsp;utilized\u0026nbsp;due to its\u0026nbsp;notable\u0026nbsp;transformation efficiency and\u0026nbsp;the\u0026nbsp;well-established\u0026nbsp;mechanisms\u0026nbsp;and\u0026nbsp;technologies associated with it\u0026nbsp;\u003csup\u003e19\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eKey\u0026nbsp;factors\u0026nbsp;influencing\u0026nbsp;efficiency in \u003cem\u003eAgrobacterium\u003c/em\u003e-mediated transformation include bacterial solution concentration,\u0026nbsp;infiltration duration, co-culture\u0026nbsp;duration, antibiotic screening, and the\u0026nbsp;choice\u0026nbsp;of explants.\u0026nbsp;Fine-tuning\u0026nbsp;these factors\u0026nbsp;and identifying optimal\u0026nbsp;thresholds\u0026nbsp;can amplify\u0026nbsp;transformation efficiency,\u0026nbsp;yet\u0026nbsp;excessive adjustments may\u0026nbsp;potentially diminish\u0026nbsp;efficiency.\u003c/p\u003e\n\u003cp\u003eVarious plant tissues or organs, such as roots, stems, leaves, somatic embryos, mature embryos, and immature embryos, can\u0026nbsp;function\u0026nbsp;as\u0026nbsp;recipients\u0026nbsp;for genetic transformation\u0026nbsp;\u003csup\u003e20\u003c/sup\u003e\u003csup\u003e21\u003c/sup\u003e. In\u0026nbsp;studies on\u0026nbsp;conifer genetic transformation, mature and immature zygotic embryos are commonly utilized\u0026nbsp;\u003csup\u003e22\u003c/sup\u003e. For\u0026nbsp;example,\u0026nbsp;\u003cem\u003ePinus massoniana\u003c/em\u003e \u003csup\u003e23\u003c/sup\u003e and\u0026nbsp;slash\u0026nbsp;pine\u0026nbsp;\u003csup\u003e24\u003c/sup\u003e have been\u0026nbsp;genetically transformed using mature zygotic embryos. Gao\u003csup\u003e9\u003c/sup\u003e employed\u0026nbsp;immature \u003cem\u003ePinus koraiensis\u003c/em\u003e zygotic embryos to induce embryonic\u0026nbsp;callus\u0026nbsp;tissue, establishing a regeneration system\u0026nbsp;via\u0026nbsp;somatic embryogenesis. In\u0026middot;\u0026nbsp;the current\u0026nbsp;study,\u0026nbsp;the\u0026nbsp;\u003cem\u003ePinus koraiensis\u003c/em\u003e embryonic callus was\u0026nbsp;selected\u0026nbsp;for genetic transformation, achieving a high transformation rate of 88.89%, with the resistant embryonic callus cultured into resistant somatic embryos.\u003c/p\u003e\n\u003cp\u003eDuring\u0026nbsp;the transformation process, both\u0026nbsp;infiltration duration\u0026nbsp;and concentration are\u0026nbsp;critical\u0026nbsp;factors for successful outcomes.\u0026nbsp;Insufficient\u0026nbsp;concentration of the bacterial solution\u0026nbsp;leads to inadequate\u0026nbsp;adherence\u0026nbsp;of \u003cem\u003eAgrobacterium\u003c/em\u003e to the plant material, resulting in low transformation efficiency. Conversely, excessively high\u0026nbsp;concentrations result in the\u0026nbsp;over-proliferation of \u003cem\u003eAgrobacterium\u003c/em\u003e within\u0026nbsp;the plant material during later co-culture stages,\u0026nbsp;posing\u0026nbsp;challenges in subsequent decolonization and\u0026nbsp;potentially\u0026nbsp;risking plant\u0026nbsp;senescence. Infiltration\u0026nbsp;duration also\u0026nbsp;plays a role in\u0026nbsp;transformation efficiency. Too short a\u0026nbsp;duration\u0026nbsp;does not\u0026nbsp;permit\u0026nbsp;sufficient attachment\u0026nbsp;of \u003cem\u003eAgrobacterium\u003c/em\u003e to the plant material, while excessively long\u0026nbsp;infiltration\u0026nbsp;periods lead to an\u0026nbsp;unnecessary\u0026nbsp;buildup of \u003cem\u003eAgrobacterium\u003c/em\u003e\u003cem\u003e\u003csup\u003e21\u003c/sup\u003e\u003c/em\u003e.\u003c/p\u003e\n\u003cp\u003eIn conifer genetic transformation systems, the bacterial solution concentrations during\u0026nbsp;infiltration\u0026nbsp;typically range\u0026nbsp;from an OD\u003csub\u003e600\u003c/sub\u003e absorbance\u0026nbsp;of 0.3\u0026nbsp;to\u0026nbsp;0.8,\u0026nbsp;with infiltration\u0026nbsp;times\u0026nbsp;ranging\u0026nbsp;from\u0026nbsp;ten\u0026nbsp;to 40\u0026nbsp;minutes. For\u0026nbsp;example, some\u0026nbsp;\u003cem\u003eLarix\u003c/em\u003e species have\u0026nbsp;achieved the highest transformation rate with a bacterial solution concentration\u0026nbsp;of approximately\u0026nbsp;OD\u003csub\u003e600\u003c/sub\u003e=0.6 and an\u0026nbsp;infiltration\u0026nbsp;time of 20\u0026nbsp;min25. Similarly, optimal results\u0026nbsp;in the genetic transformation of \u003cem\u003ePinus bungeana\u003c/em\u003e were observed\u0026nbsp;with an OD\u003csub\u003e600\u003c/sub\u003e=0.4 and an\u0026nbsp;infiltration\u0026nbsp;time of 30\u0026nbsp;min\u0026nbsp;26.\u0026nbsp;Transformation of\u0026nbsp;\u003cem\u003ePinus taeda\u003c/em\u003e exhibited a transient expression rate of\u0026nbsp;around\u0026nbsp;70% with a bacteriophage concentration OD\u003csub\u003e600\u003c/sub\u003e=0.8 and\u0026nbsp;infiltration\u0026nbsp;times ranging from 15 to 30\u0026nbsp;min\u0026nbsp;\u003csup\u003e27\u003c/sup\u003e. In this study, the most favorable outcomes were\u0026nbsp;attained\u0026nbsp;by\u0026nbsp;using\u0026nbsp;an OD\u003csub\u003e600\u003c/sub\u003e absorbance of 0.5 for infiltrating \u003cem\u003ePinus koraiensis\u003c/em\u003e embryonic callus\u0026nbsp;for 20 min.\u003c/p\u003e\n\u003cp\u003eThe co-culture\u0026nbsp;phase signifies\u0026nbsp;the stage at which T-DNA integration is\u0026nbsp;conveyed\u0026nbsp;into the plant genome. Optimal co-culture conditions for conifers\u0026nbsp;have been determined to be two to four\u0026nbsp;days in dark culture at 25\u0026deg;C.\u0026nbsp;Straying\u0026nbsp;from this timeframe, either too short or too long, can diminish conversion efficiency\u0026nbsp;\u003csup\u003e28\u003c/sup\u003e. For\u0026nbsp;example, in the genetic transformation of\u0026nbsp;the\u0026nbsp;embryonic callus of\u003cem\u003e\u0026nbsp;\u003c/em\u003ehybrid fir\u0026nbsp;\u003csup\u003e29\u003c/sup\u003e and white spruce\u0026nbsp;\u003csup\u003e30\u003c/sup\u003e, the co-culture\u0026nbsp;period\u0026nbsp;was\u0026nbsp;fixed\u0026nbsp;at\u0026nbsp;two\u0026nbsp;days.\u0026nbsp;For\u0026nbsp;mature\u0026nbsp;zygotic embryos of \u003cem\u003ePinus massoniana\u003c/em\u003e\u003csup\u003e\u003cem\u003e23\u003c/em\u003e\u003c/sup\u003e, incubation\u0026nbsp;spanned\u0026nbsp;a total of\u0026nbsp;three\u0026nbsp;days. The results of\u0026nbsp;this\u0026nbsp;study\u0026nbsp;indicated\u0026nbsp;that a\u0026nbsp;two-day co-culture\u0026nbsp;duration\u0026nbsp;yielded\u0026nbsp;superior\u0026nbsp;efficiency in tissue transformation compared to\u0026nbsp;one, three, or four\u0026nbsp;days. Prolonged\u0026nbsp;co-culture beyond\u0026nbsp;three\u0026nbsp;days\u0026nbsp;led to repetitive\u0026nbsp;decolonization of tissues due to\u0026nbsp;elevated\u0026nbsp;bacterial\u0026nbsp;concentrations, ultimately leading to\u0026nbsp;tissue\u0026nbsp;death.\u003c/p\u003e\n\u003cp\u003eThe\u0026nbsp;selection\u0026nbsp;of antibiotic type and its concentration are\u0026nbsp;crucial\u0026nbsp;factors\u0026nbsp;that influence\u0026nbsp;the transformation process. Sensitivity tests\u0026nbsp;conducted\u0026nbsp;on other plant\u0026nbsp;species have\u0026nbsp;revealed that employing multiple screenings\u0026nbsp;enhances\u0026nbsp;the transformation rate\u0026nbsp;while reducing\u0026nbsp;the false positive rate. In\u0026nbsp;conifer\u0026nbsp;genetic transformation, two primary antibiotics, Kan and Hyg,\u0026nbsp;are\u0026nbsp;predominantly\u0026nbsp;utilized\u0026nbsp;for screening.\u003cem\u003e\u0026nbsp;\u003c/em\u003eYaupon\u0026nbsp;\u003csup\u003e31\u003c/sup\u003e achieved resistance in\u0026nbsp;tissues\u0026nbsp;by conducting\u0026nbsp;Kan screening at concentrations ranging from 50 to 75 mg\u0026middot;L\u003csup\u003e-1\u003c/sup\u003e.\u0026nbsp;A\u0026nbsp;positive rate of 66.2% in obtaining resistant embryogenic callus\u0026nbsp;was achieved in\u0026nbsp;hybrid larch under three consecutive screens with 20 mg\u0026middot;L\u003csup\u003e-1\u003c/sup\u003e of Kan\u0026nbsp;\u003csup\u003e32\u003c/sup\u003e. The false positive rate of resistance obtained\u0026nbsp;via\u0026nbsp;Hyg screening in \u003cem\u003eLarix olgensis\u003c/em\u003e was only 11.66%\u003csup\u003e33\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eIn this study, sensitivity experiments were\u0026nbsp;carried out\u0026nbsp;on the \u003cem\u003ePinus koraiensis\u003c/em\u003e embryonic\u0026nbsp;callus, demonstrating its\u0026nbsp;insensitivity to Kan. At a Kan concentration of 100 mg\u0026middot;L\u003csup\u003e-1\u003c/sup\u003e, significant\u0026nbsp;differences\u0026nbsp;in the proliferation rate of \u003cem\u003ePinus koraiensis\u003c/em\u003e embryonic callus\u0026nbsp;were not detected.\u0026nbsp;Conversely,\u0026nbsp;when\u0026nbsp;screening with Hyg as the antibiotic, the\u0026nbsp;proliferation rate of \u003cem\u003ePinus koraiensis\u003c/em\u003e embryonic callus\u0026nbsp;significantly decreased. At\u0026nbsp;a concentration of 4 mg\u0026middot;L\u003csup\u003e-1\u003c/sup\u003e,\u0026nbsp;the proliferation rate was 68.42%,\u0026nbsp;and the embryonic callus\u0026nbsp;barely\u0026nbsp;proliferated at concentrations\u0026nbsp;surpassing\u0026nbsp;20 mg\u0026middot;L\u003csup\u003e-1\u003c/sup\u003e, with the group tissues\u0026nbsp;dying\u0026nbsp;at 40 mg\u0026middot;L\u003csup\u003e-1\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eFurthermore, the number of screenings was hypothesized to affect the detected positive conversion rate. Due to the sensitivity of embryonic callus, the initial screening yielded fewer resistant tissues and a higher false positive rate. Subsequent screenings, particularly the second and third rounds, effectively decreased the false positive rate and enhanced the conversion rate. Consequently, 36 resistants embryonic calli were obtained in this study using Hyg as the antibiotic, subjected to three consecutive screenings lasting 21 days each, resulting in a significantly low false positive rate of 11.11%.\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eIn this study, a highly efficient genetic transformation system for \u003cem\u003ePinus koraiensis\u003c/em\u003e embryonic callus was successfully established and optimized, marking a significant breakthrough in this field. \u003cem\u003eAgrobacterium\u003c/em\u003e strain GV3101 was utilized in combination with the binary vector VB191103-1905rcy, resulting in the acquisition of 36 resistant embryogenic calli under specific conditions: an OD\u003csub\u003e600\u003c/sub\u003e absorbance value of 0.6 for the infiltrating solution, a two-day co-culture duration, and 10 mg\u0026middot;L\u003csup\u003e-1\u003c/sup\u003e Hyg as the screening antibiotic. The transformation rate reached an impressive 88.89%, as validated by GUS and PCR analyses. This innovative study not only introduced a new method for investigating the gene function of \u003cem\u003ePinus koraiensis\u003c/em\u003e but also established a crucial foundation for developing a regenerative genetic transformation system for \u003cem\u003ePinus koraiensis\u003c/em\u003e plants.\u003c/p\u003e"},{"header":"Methods","content":"\u003cp\u003e\u003cstrong\u003ePlant material\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eLing \u003csup\u003e34\u003c/sup\u003e provided \u003cem\u003ePinus koraiensis\u003c/em\u003e embryonic material, which underwent induction and was then inoculated in a DCR medium (Supplementary File 1). This medium consisted of 0.5 mg\u0026middot;L\u003csup\u003e-1\u003c/sup\u003e 2,4-dichlorophenoxyacetic acid (2,4-D), 0.1 mg\u0026middot;L\u003csup\u003e-1\u003c/sup\u003e 6-benzyladenine (6-BA), 30 g\u0026middot;L\u003csup\u003e-1\u003c/sup\u003e sucrose, 4 g\u0026middot;L\u003csup\u003e-1\u003c/sup\u003e gellan gum, 0.1 mg\u0026middot;L\u003csup\u003e-1\u003c/sup\u003e inositol, 0.5 mg\u0026middot;L\u003csup\u003e-1\u003c/sup\u003e glutamine (Gln), 0.5 mg\u0026middot;L\u003csup\u003e-1\u003c/sup\u003e casein hydrolyzed (CH), with a pH of 5.9. Cultivation occurred in darkness at 25\u0026deg;C, with reproductive successions carried out every two days.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAgrobacterium strains\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe \u003cem\u003eAgrobacterium\u003c/em\u003e strain (GV3101) and the plasmid (pBI121) used in this study were maintained in the laboratory. The vector strain utilized was VB191103-1905rcy, with the plasmid, it carried containing the \u0026beta;-glucosidase gene (GUS), sourced from the State Key Laboratory of Forest Genetic Breeding, Northeast Forestry University, China.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAntibiotic sensitivity test of embryonic callus\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFollowing a ten-day\u0026nbsp;incubation in\u0026nbsp;the\u0026nbsp;DCR proliferation medium,\u0026nbsp;about\u0026nbsp;0.2 g of embryonic callus was transferred to\u0026nbsp;the\u0026nbsp;DCR medium\u0026nbsp;supplemented with\u0026nbsp;0.5 mg\u0026middot;L\u003csup\u003e-1\u003c/sup\u003e 2,4-D, 0.1 mg\u0026middot;L\u003csup\u003e-1\u003c/sup\u003e 6-BA, and 30 g\u0026middot;L\u003csup\u003e-1\u003c/sup\u003e sucrose. The medium also contained additional components such as 4 g\u0026middot;L\u003csup\u003e-1\u003c/sup\u003e gellan gum, 0.1 mg\u0026middot;L\u003csup\u003e-1\u003c/sup\u003e inositol, 0.5 mg\u0026middot;L\u003csup\u003e-1\u003c/sup\u003e Gln, 0.5 mg\u0026middot;L\u003csup\u003e-1\u003c/sup\u003e casein hydrolyzed, along with cefotaxime (Cef), kanamycin (Kan), or hygromycin (Hyg), at pH 5.9. Screening was conducted at 25\u0026plusmn;1\u0026deg;C for 15 days under dark conditions. The fresh mass of the embryonic callus was measured, and this process was repeated three times. Gradient concentrations were employed for Kan (0, 10, 20, 30, and 40 mg\u0026middot;L\u003csup\u003e-1\u003c/sup\u003e), Hyg (0, 4, 8, 10, 20, 40, and 60 mg\u0026middot;L\u003csup\u003e-1\u003c/sup\u003e), and Cef (0, 100, 200, 300, and 400 mg\u0026middot;L\u003csup\u003e-1\u003c/sup\u003e).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eAgrobacterium\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;strain culture\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA small volume of bacterial solution was drawn\u0026nbsp;up\u0026nbsp;into an inoculation loop and streaked onto\u0026nbsp;solid\u0026nbsp;yeast extract mannitol broth medium (YEB)\u0026nbsp;supplemented with\u0026nbsp;50 mg\u0026middot;L\u003csup\u003e-1\u003c/sup\u003e Kan, Hyg, and Rifampicin (Rif), then cultured for two days at 28℃. Following this, single colonies were selected and transferred into 20 mL of liquid YEB medium with 20 mg\u0026middot;L\u003csup\u003e-1\u003c/sup\u003e Kan and Rif, shaken at 200 r\u0026middot;min\u003csup\u003e-1\u003c/sup\u003e, and cultured at 28℃ for 16-18 hours in\u0026nbsp;darkness\u0026nbsp;until reaching an\u0026nbsp;optical density (OD\u003csub\u003e600\u003c/sub\u003e)\u0026nbsp;of 0.8 to 1.0. Subsequently, 1 mL of the bacterial\u0026nbsp;culture\u0026nbsp;was inoculated into\u0026nbsp;a\u0026nbsp;liquid\u0026nbsp;YEB\u0026nbsp;medium\u0026nbsp;containing 50 mg\u0026middot;L\u003csup\u003e-1\u003c/sup\u003e Kan and Rif for five to eight hours, allowing the \u003cem\u003eAgrobacterium\u003c/em\u003e to attain the logarithmic growth phase with an OD\u003csub\u003e600\u003c/sub\u003e of 0.6 to 0.8.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eGenetic transformation\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe fresh bacterial solution was transferred into a sterile centrifuge tube and\u0026nbsp;centrifuged\u0026nbsp;(Therom, China) at 4℃ and\u0026nbsp;8,000\u0026nbsp;rpm for\u0026nbsp;ten minutes\u0026nbsp;to collect the bacterial pellet for the\u0026nbsp;infiltration\u0026nbsp;solution. Fresh embryonic callus was carefully\u0026nbsp;chosen\u0026nbsp;and\u0026nbsp;submerged\u0026nbsp;in the\u0026nbsp;infiltration\u0026nbsp;solution, which\u0026nbsp;contained\u0026nbsp;100 \u0026micro;M\u0026middot;L\u003csup\u003e-1\u003c/sup\u003e acetosyringone\u0026nbsp;(AS). The concentration of the\u0026nbsp;infiltration\u0026nbsp;solution was adjusted to absorbance values of 0.4, 0.6, or 1.0 at OD\u003csub\u003e600\u003c/sub\u003e.\u0026nbsp;After a 20-min infiltration\u0026nbsp;period, the solution was\u0026nbsp;poured off, and the tissue surface was gently blotted with sterile filter paper before being transferred to the co-culture medium (DCR+250 mg\u0026middot;L\u003csup\u003e-1\u003c/sup\u003e Cef) for\u0026nbsp;two, three, or\u0026nbsp;four\u0026nbsp;days at 25\u0026plusmn;1 ℃ in\u0026nbsp;the\u0026nbsp;dark.\u003c/p\u003e\n\u003cp\u003eFollowing\u0026nbsp;co-culture, the tissues\u0026nbsp;were rinsed twice\u0026nbsp;with sterile water for two\u0026nbsp;minutes each, followed by two washes with a suspension containing 500 mg\u0026middot;L\u003csup\u003e-1\u003c/sup\u003e Cef for 3 min each. Any excess water on the surface of the cleaned callus was carefully blotted dry with sterile filter paper. The embryonic callus was spread out into sheets on a recovery medium at 25\u0026plusmn;1\u0026deg;C and cultured in the dark for seven, 14, or 21 days. Following this, the resistant tissues underwent screening using a specialized screening medium. The number of resistant tissues obtained was documented over three successive screenings, each lasting 21 days. These resistant tissues were then subjected to GUS histochemical staining and PCR analysis at the molecular level. Once appropriately confirmed as resistant, the embryonic callus underwent succession culture and somatic embryo maturation (Figure 6).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eGUS histochemical assay\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe resistant tissues were carefully selected and\u0026nbsp;immersed\u0026nbsp;in an appropriate volume of GUS staining solution, formulated with 100 mM sodium phosphate buffer (pH=7.0), 0.5 mg\u0026middot;mL\u003csup\u003e-1\u0026nbsp;\u003c/sup\u003eX-Gluc (5-bromo-4-chloro-3-indolyl-\u0026beta;-D-glucuronide), 1% Triton X-100, 1% DMSO, and 10 mM EDTA. The staining solution was infiltrated using a vacuum pump until no bubbles were visible. The embryogenic callus underwent staining in a water bath at 37℃ for one week. Non-transformed tissues were used as the negative control, and the color development status of the embryonic callus was observed and documented. Among these, tissues displaying a blue-green color were identified as transgenic tissues, whereas colorless transparent or slightly yellow tissues were considered false-positive tissues.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePCR analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eUsing \u003cem\u003ePinus koraiensis\u003c/em\u003e resistant callus DNA as a template, genomic DNA was extracted from 14 randomly selected resistant cell lines employing the CTAB method. PCR molecular assays were carried out with upstream and downstream primers for the target genes. The upstream primer sequence was 5\u0026apos;-CAAAGCAAGTGGATTGATGTGAT-3\u0026apos;, and the downstream primer sequence was 5\u0026apos;-AGAGAGAAAAGGGTCCTAACCAAGA-3\u0026apos;. The reaction mixture consisted of 10\u0026micro;L of Green Taq MIX, 1\u0026micro;L each of upstream and downstream primers, 1\u0026micro;L of DNA, and 7\u0026micro;L of ddH\u003csub\u003e2\u003c/sub\u003eO. The reaction conditions\u0026nbsp;included\u0026nbsp;denaturation at 94℃ for 3 min,\u0026nbsp;followed by\u0026nbsp;denaturation at 94℃ for 30 sec, annealing at 60℃ for 15 sec, extension at 72℃ for 15 sec, repeated for 35 cycles.\u0026nbsp;The final\u0026nbsp;extension\u0026nbsp;was\u0026nbsp;at 72℃ for 10 min.\u003c/p\u003e\n\u003cp\u003eThe PCR samples were then separated using a 1.0% (w/v) agarose gel electrophoresis with wild-type \u003cem\u003ePinus koraiensis\u003c/em\u003e embryonic callus as the negative control and \u003cem\u003eAgrobacterium\u003c/em\u003e solution as the positive control. The electrophoresis results were observed and captured using a gel imager (Tanon 2500R, China). The resistant embryogenic calli were identified based on the presence of correct destination bands.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSomatic embryo maturation\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe approach for somatic embryo maturation was adapted from Peng \u003csup\u003e35\u003c/sup\u003e with some changes. Three grams of various transgenic \u003cem\u003ePinus koraiensis\u003c/em\u003e embryonic callus samples were placed into 20 mL of liquid medium (DCR+30 g\u0026middot;L\u003csup\u003e-1\u003c/sup\u003e sucrose) devoid of any hormones and incubated for seven days. Subsequently, the mixture was poured off and excess liquid was absorbed using sterile filter paper. The embryonic callus was then dried on an ultra-clean bench for ten minutes.\u003c/p\u003e\n\u003cp\u003eThe transgenic \u003cem\u003ePinus koraiensis\u003c/em\u003e embryogenic callus obtained through the aforementioned methods was transferred to a DCR maturation medium, comprising varying concentrations of gellan gum (8, 10, and 12 g\u0026middot;L\u003csup\u003e-1\u003c/sup\u003e), 20 mg\u0026middot;L\u003csup\u003e-1\u003c/sup\u003e ABA, 0.1 g\u0026middot;L\u003csup\u003e-1\u003c/sup\u003e inositol, 0.5 g\u0026middot;L\u003csup\u003e-1\u003c/sup\u003e Gln, 0.5 g\u0026middot;L\u003csup\u003e-1\u003c/sup\u003e CH, 30 g\u0026middot;L\u003csup\u003e-1\u003c/sup\u003e maltose, with pH adjusted to 5.9. All somatic embryogenesis events were tallied after a 10-week period.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStatistical analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSPSS statistical analysis software was used for variance analysis, setting the significance level at p \u0026lt; 0.05. The tables and figures were generated using Microsoft Excel 2010.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eData Availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll data in this study are available in the manuscript or the Supplementary materials.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthical\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003edeclarations\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe plant collection and use was in accordance with all the relevant guidelines.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by Scientific and Technological Innovation 2030 -Major Project of Agricultural Biological Breeding (No.2023ZD040580204). The National Key Research and Development Program of China (No.2023YFD2200605).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eS.L. conceived and designed the experiments; H.Z. provided the financial support; L.Y. and H.S. provided the \u003cem\u003ePinus koraiensis\u003c/em\u003e embryonic callus; H.H. and H.D. performed the experiments, analyzed the data, prepared the figures and tables, and they contributed equally to this work; Y.W and W.Z. reviewed drafts of the paper. All authors have read and agreed to the published version of the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing interests\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eZhang Z, Zhang HG, Mo C, Zhang L. Transcriptome Sequencing Analysis and Development of EST\u0026middot;SSR Markers for \u003cem\u003ePinus koraiensis\u003c/em\u003e. \u003cem\u003eScientia Silvae Sinicae\u003c/em\u003e. 51:114-120 (2015)\u003c/li\u003e\n\u003cli\u003eHuang YY, Zhu XQ, Zhu Y, Wang ZY. \u003cem\u003ePinus koraiensis\u003c/em\u003e polyphenols: structural identification, in vitro antioxidant activity, immune function and inhibition of cancer cell proliferation. \u003cem\u003eFood Funct\u003c/em\u003e. 12(9):4176-4198, Doi:10.1039/d0fo03347b (2021).\u003c/li\u003e\n\u003cli\u003ePullman, GS, Bucalo K. \u003cem\u003ePine somatic\u003c/em\u003e embryogenesis using zygotic embryos as explants. \u003cem\u003eMethods Mol. Biol.\u003c/em\u003e 710:267-91, Doi:10.1007/978-1-61737-988-8-9 (2011).\u003c/li\u003e\n\u003cli\u003eGao F, Peng CX, Wang H, Tretyakova IN, Nosov AM, Shen HL, Yang L. Key Techniques for Somatic Embryogenesis and Plant Regeneration of \u003cem\u003ePinus koraiensis\u003c/em\u003e. Forests. 111:9, Doi:10.3390/f11090912 (2020).\u003c/li\u003e\n\u003cli\u003eGao F, Peng CX, Zhang Y, Wang H, Shen HL, Yang L. Glutathione Plays a Positive Role in the Proliferation of \u003cem\u003ePinus koraiensis\u003c/em\u003e Embryogenic Cells. \u003cem\u003eInt. J. Mol. Sci.\u003c/em\u003e 23(23):14679, Doi:10.3390/ijms232314679 (2022).\u003c/li\u003e\n\u003cli\u003ePeng CX, Gao F, Wang H, Tretyakova IN, Nosov AM, Shen HL, Yang L. Morphological and Physiological Indicators for Screening Cell Lines with High Potential for Somatic Embryo Maturation at an Early Stage of Somatic Embryogenesis in \u003cem\u003ePinus Koraiensis\u003c/em\u003e. \u003cem\u003ePlants Basel\u003c/em\u003e. 11(14):1867. doi:10.3390/plants11141867 (2022).\u003c/li\u003e\n\u003cli\u003ePeng CX, Gao F, Wang H, Shen HL, Yang L. Optimization of maturation process for somatic embryo production and cryopreservation of embryogenic tissue in \u003cem\u003ePinus koraiensis\u003c/em\u003e. \u003cem\u003ePlant Cell Tissue and Organ Culture\u003c/em\u003e. 1144:1, Doi:10.1007/s11240-020-01918-y (2020).\u003c/li\u003e\n\u003cli\u003ePeng CX, Gao F, Tretyakova IN, Nosov AM, Shen HL, Yang L. Transcriptomic and Metabolomic Analysis of \u003cem\u003eKorean pine\u003c/em\u003e Cell Lines with Different Somatic Embryogenic Potential. \u003cem\u003eInt. J. Mol. Sci\u003c/em\u003e. 23(21):13301, Doi:10.3390/ijms232113301 (2022).\u003c/li\u003e\n\u003cli\u003eGao F, Shi YJ, Wang RR, Tretyakova IN, Nosov AM, Shen HL, Yang L. Exogenous Glutathione Promotes the Proliferation of \u003cem\u003ePinus koraiensis\u003c/em\u003e Embryonic Cells and the Synthesis of Glutathione and Ascorbic Acid. \u003cem\u003ePlants\u003c/em\u003e. 11:2586-2586, Doi:10.1007/s11240-022-02348-8 (2022).\u003c/li\u003e\n\u003cli\u003eCastander Olarieta A, Moncalean P, Montalban IA. Somatic Embryogenesis in \u003cem\u003ePines\u003c/em\u003e. \u003cem\u003eMethods Mol. Biol.\u003c/em\u003e 25:41-56, Doi:10.1007/978-1-0716-2485-2_4 (2022).\u003c/li\u003e\n\u003cli\u003eRamkumar TR, Lenka SK, Arya SS, Bansal KC. A Short History and Perspectives on Plant Genetic Transformation. \u003cem\u003eMethods Mol. Biol.\u003c/em\u003e 2124:39-68, Doi:10.1007/978-1-0716-0356-7_3 (2020).\u003c/li\u003e\n\u003cli\u003eHuang Y, Diner AM, Karnosky DF. \u003cem\u003eAgrobacterium\u003c/em\u003e rhizogenes-mediated genetic transformation and regeneration of a conifer: \u003cem\u003eLarix decidua\u003c/em\u003e. \u003cem\u003eIn\u003c/em\u003e\u003cem\u003eVitro Cellular \u0026amp; Developmental Biology\u003c/em\u003e-\u003cem\u003ePlant\u003c/em\u003e. 27:201-7 (1991).\u003c/li\u003e\n\u003cli\u003eMaruyama TE, Hosoi Y. Progress in Somatic Embryogenesis of \u003cem\u003eJapanese Pines\u003c/em\u003e. \u003cem\u003eFront Plant Sci\u003c/em\u003e. 10:31, Doi:10.3389/fpls.2019.00031 (2019).\u003c/li\u003e\n\u003cli\u003eDang SF, Zhang LF, Han SY, Qi LW. \u003cem\u003eAgrobacterium\u003c/em\u003e-Mediated Genetic Transformation of \u003cem\u003eLarix kaempferi\u003c/em\u003e (\u003cem\u003eLamb.\u003c/em\u003e) \u003cem\u003eCarr\u003c/em\u003e. Embryogenic Cell Suspension Cultures and Expression Analysis of Exogenous Genes. \u003cem\u003eForests\u003c/em\u003e. 13(9):1436-1436, Doi:10.3390/f13091436 (2022).\u003c/li\u003e\n\u003cli\u003eTang W. (2001). \u003cem\u003eAgrobacterium\u003c/em\u003e-mediated transformation and assessment of factors influencing transgene expression in loblolly pine (Pinus taeda L.). Cell\u003cem\u003e Research\u003c/em\u003e. 11(3):237-43, Doi:10.1038/sj.cr.7290092 (2001).\u003c/li\u003e\n\u003cli\u003eCui Y, Zhao J, Gao Y, Zhao R, Zhang J, Kong L. Efficient Multi-Sites Genome Editing and Plant Regeneration via Somatic Embryogenesis in \u003cem\u003ePicea glauca\u003c/em\u003e. \u003cem\u003eFront Plant Sci\u003c/em\u003e. 12:751-891, Doi:10.3389/fpls.2021.751891 (2021).\u003c/li\u003e\n\u003cli\u003eHazubska-Przybyl T, Wawrzyniak MK, Kijowska-Oberc J, Staszak AM, RRatajczak E. Somatic Embryogenesis of \u003cem\u003eNorway Spruce\u003c/em\u003e and \u003cem\u003eScots Pine\u003c/em\u003e: Possibility of Application in Modern Forestry. \u003cem\u003eForests. \u003c/em\u003e13:2, Doi:10.3390/f13020155 (2022).\u003c/li\u003e\n\u003cli\u003eLiang Y, Xu X, Shen HL, Gao ML, Zhao Y, Bai X. Morphological and endogenous phytohormone changes during long-term embryogenic cultures in \u003cem\u003eKorean pine\u003c/em\u003e. \u003cem\u003ePlant Cell Tissue and Organ Culture\u003c/em\u003e. 151:253-264, Doi:10.1007/s11240-022-02348-8 (2022).\u003c/li\u003e\n\u003cli\u003ePeng CX, Gao F, Wang H, Shen HL, Yang L. Physiological and Biochemical Traits in \u003cem\u003eKorean pine\u003c/em\u003e Somatic Embryogenesis. \u003cem\u003eForests\u003c/em\u003e. 11(5): 577-577, Doi10.3390/f1105057 (2020).\u003c/li\u003e\n\u003cli\u003eWang B, Zhang Z, Yin Z, Feng C, Wang Q. Novel and potential application of cryopreservation to plant genetic transformation. \u003cem\u003eBiotechnol. Adv\u003c/em\u003e. 30(3):604-12, Doi:10.1016/j. biotechadv.2011.10.008 (2011).\u003c/li\u003e\n\u003cli\u003eYao R, Wang Y. An advanced protocol for the establishment of plantlets originating from somatic embryos in \u003cem\u003ePinus massoniana\u003c/em\u003e. \u003cem\u003e3\u003c/em\u003e\u003cem\u003eBiotech\u003c/em\u003e. 10(9):394, Doi:10.1007/s13205-020-02385-0 (2020).\u003c/li\u003e\n\u003cli\u003eZhang Y. Establishment of genetic transformation receptor systems and \u003cem\u003eAgrobacterium\u003c/em\u003e-mediated genetic transformation in three coniferous species. Dissertation for the Degree. Shanghai: Graduate School of Chinese Academy of Sciences (2006).\u003c/li\u003e\n\u003cli\u003eMaleki SS, Mohammadi K, Ji KS. Study on factors influencing transformation efficiency in \u003cem\u003ePinus massoniana\u003c/em\u003e using \u003cem\u003eAgrobacterium\u003c/em\u003e tumefaciens. \u003cem\u003ePlant Cell Tissue and Organ Culture.\u003c/em\u003e 133(3):437-445, Doi:10.1007/s11240-018-1388-7 (2018).\u003c/li\u003e\n\u003cli\u003eTang. W, Xiao B, Fei YJ. \u003cem\u003eSlash pine\u003c/em\u003e genetic transformation through embryo cocultivation with a tumefaciens and transgenic plant regeneratio. I\u003cem\u003en Vitro Cellular \u0026amp; Developmental Biology. Plant\u003c/em\u003e. 50(2): 199-209 (2014).\u003c/li\u003e\n\u003cli\u003eLin XF, Zhang WB, Takechi K, Takio S, Ono, Takano H. Stable genetic transformation of \u003cem\u003eLarix gmelinii\u003c/em\u003e L. by particle bombardment of zygotic embryos. \u003cem\u003ePlant Cell Rep\u003c/em\u003e. 24:418-425, Doi:10.1007/s00299-005-0955-7 (2005).\u003c/li\u003e\n\u003cli\u003eZhang L, Qi LW, Han SY. Differentially expressed genes during \u003cem\u003eLarix\u003c/em\u003e somatic embryo maturation and the expression profile of partial genes. \u003cem\u003eYi chuan Hereditas\u003c/em\u003e. 31:540-5 (2009).\u003c/li\u003e\n\u003cli\u003eTang W, Newton RJ. Genetic transformation of conifers and its application in forest biotechnology. \u003cem\u003ePlant cell Reports\u003c/em\u003e. 22(1):1-15, Doi: 10.1007/s00299-003-0670-1 (2003).\u003c/li\u003e\n\u003cli\u003eRamkumar TR, Lenka SK, Arya SS, Bansal KC. A Short History and Perspectives on Plant Genetic Transformation. \u003cem\u003eMethods Mol Biol\u003c/em\u003e. 24:39-68, Doi:10.1007/978-1-0716-0356-7_3 (2020).\u003c/li\u003e\n\u003cli\u003eSalaj T, Moravcikova J, Vookova B, Salaj J. \u003cem\u003eAgrobacterium\u003c/em\u003e-mediated transformation of embryogenic tissues of \u003cem\u003ehybrid firs\u003c/em\u003e (\u003cem\u003eAbies spp\u003c/em\u003e.) and regeneration of transgenic emblings. \u003cem\u003eBiotechnology letters\u003c/em\u003e. 331:647-652, Doi:10.1007/s10529-009-9923-6 (2009).\u003c/li\u003e\n\u003cli\u003eLe VQ, Belles-Isles J, Dusabenyagasani M, Trembiay FM. An improved procedure for production of \u003cem\u003ewhite spruce\u003c/em\u003e (\u003cem\u003ePicea glauca\u003c/em\u003e) transgenic plants using \u003cem\u003eAgrobacterium\u003c/em\u003e tumefaciens. \u003cem\u003eJournal of experimental botany\u003c/em\u003e. 52:2089-2095, Doi:10.1093/jexbot/52.364.2089 (2001).\u003c/li\u003e\n\u003cli\u003eLiu S, Ma J, Liu H, Guo Y, Li W, Niu S. An efficient system for \u003cem\u003eAgrobacterium\u003c/em\u003e-mediated transient transformation in Pinus tabuliformis. Plant Methods. 16:52, Doi:10.1186/s13007-020-00594-5 (2020).\u003c/li\u003e\n\u003cli\u003eGleeson D, Lelu-Walter M, Parkinson M. Overproduction of proline in transgenic hybrid larch (Larix x leptoeuropaea (Dengler)) cultures renders them tolerant to cold, salt and frost. Molecular Breeding, 15:21-29, Doi:10.1007/s11032-004-1363-3 (2005).\u003c/li\u003e\n\u003cli\u003eSong Y. et al. Stable and Efficient \u003cem\u003eAgrobacterium\u003c/em\u003e-Mediated Genetic Transformation of \u003cem\u003eLarch\u003c/em\u003e Using Embryogenic Callus.\u003cem\u003e Frontiers in Plant Science.\u003c/em\u003e 11: 84492-584492, Doi:10.3389/fpls.2020.584492 (2020).\u003c/li\u003e\n\u003cli\u003eGao F, Peng CX, Wang H, Shen HL, Yang L. Selection of culture conditions for callus induction and proliferation by somatic embryogenesis of \u003cem\u003ePinus koraiensi\u003c/em\u003e. \u003cem\u003eJournal of Forestry Researc\u003c/em\u003eh. 32(02):483-491, Doi10.1007/s11676-020-01147-1 (2021).\u003c/li\u003e\n\u003cli\u003ePeng CX, Gao F, Wang H, Tretyakova IN, Nosov AM, Shen H.L, Yang L. Suspension Culture and Somatic Embryogenesis of \u003cem\u003eKorean pine\u003c/em\u003e. Phyton. \u003cem\u003eInt. J. Exp. Bot.\u003c/em\u003e 91:223-238, Doi:10.32604/phyton.2022.015523 (2022).\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Pinus koraiensis, Embryogenic callus, Agrobacterium-Mediated, Genetic transformation, Somatic","lastPublishedDoi":"10.21203/rs.3.rs-4173927/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4173927/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"Pinus koraiensis holds the designation of a second-class protected wild plant in China, valued for its significant economic and ecological importance. However, the lack of a well-established genetic transformation system has hindered progress in functional research and breeding applications for this species. This study aimed to establish a reliable and efficient genetic transformation system of Pinus koraiensis embryonic callus, building upon somatic embryogenesis technology. Utilizing Pinus koraiensis embryonic callus and GUS as the reporter gene, Agrobacterium-mediated transformation was employed to investigate key transformation factors, such as antibiotic type and concentration, Agrobacterium bacterial solution concentration, infiltration, and co-cultivation times. The results revealed that 10 mg·L-1 of Hygromycin (Hyg) significantly inhibited Pinus koraiensis embryonic callus proliferation, while an OD600 absorbance value of 0.6 during transformation led to a remarkable 93.42±2.13% efficiency. Optimal co-cultivation for two days resulted in a transformation rate of 82.61%, with a high GUS staining rate of 88.89% in the resistant embryonic callus. Following the optimized protocol, resistant somatic embryos were successfully obtained. This research contributes to the advancement of seed resource breeding and genetic enhancement for Pinus koraiensis, providing a solid foundation for investigating gene functions related to this species.","manuscriptTitle":"A method for permanent genetic transformation using embryonic callus of Pinus koraiensis","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-04-12 14:24:07","doi":"10.21203/rs.3.rs-4173927/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"259afeaa-30f9-4f76-827f-99ccfdbfa558","owner":[],"postedDate":"April 12th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":30560514,"name":"Biological sciences/Biotechnology/Plant biotechnology"},{"id":30560515,"name":"Biological sciences/Genetics/Plant breeding"}],"tags":[],"updatedAt":"2024-10-18T07:08:53+00:00","versionOfRecord":[],"versionCreatedAt":"2024-04-12 14:24:07","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-4173927","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4173927","identity":"rs-4173927","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}