Acetosyringone Concentration Research Articles

Improved Protocol for Efficient Agrobacterium-Mediated Transient Gene Expression in Medicago sativa L.

Medicago sativa L. (Alfalfa) is a globally recognized forage legume that has recently gained attention for its high protein content, making it suitable for both human and animal consumption. However, due to its perennial nature and autotetraploid genetics, conventional plant breeding requires a longer timeframe compared to other crops. Therefore, genetic engineering offers a faster route for trait modification and improvement. Here, we describe a protocol for achieving efficient transient gene expression in alfalfa through genetic transformation with the Agrobacterium tumefaciens pCAMBIA1304 vector. This vector contains the reporter genes β-glucuronidase (GUS) and green fluorescent protein (GFP), along with a selectable hygromycin B phosphotransferase gene, all driven by the CaMV 35s promoter. Various transformation parameters—such as different explant types, leaf ages, leaf sizes, wounding types, bacterial concentrations (OD600nm), tissue preculture periods, infection periods, co-cultivation periods, and different concentrations of acetosyringone, silver nitrate, and calcium chloride—were optimized using 3-week-old in vitro-grown plantlets. Results were attained from data based on the semi-quantitative observation of the percentage and number of GUS spots on different days of agro-infection in alfalfa explants. The highest percentage of GUS positivity (76.2%) was observed in 3-week-old, scalpel-wounded, segmented alfalfa leaf explants after 3 days of agro-infection at a bacterial concentration of 0.6, with 2 days of preculture, 30 min of co-cultivation, and the addition of 150 µM acetosyringone, 4 mM calcium chloride, and 75 µM silver nitrate. The transient expression of genes of interest was confirmed via histochemical GUS and GFP assays. The results based on transient reporter gene expression suggest that various factors influence T-DNA delivery in the Agrobacterium-mediated transformation of alfalfa. The improved protocol can be used in stable transformation techniques for alfalfa.

Establishment of Genetic Transformation System in Lilium pumilum and Functional Analysis of LpNAC6 on Abiotic Stress

Lilium pumilum is widely distributed in northeast Asia. It exhibits strong resistance and possesses high ornamental value. However, it currently lacks an efficient and stable transformation system. Therefore, we aimed to establish an effective genetic transformation system using the Agrobacterium-mediated method for L. pumilum, enabling gene transfer into the plant for gene function research and genetic engineering breeding. Our genetic transformation system achieved a maximum transformation efficiency of 7.25% under specific conditions: a kanamycin (Kana) concentration of 120mg/L, 3 days of pre-cultivation, an A. tumefaciens concentration of 0.7 OD600, an acetosyringone (AS) concentration of 20mg/L, and a 15-minute infection period. We investigated the function of the LpNAC6 from L. pumilum by observing phenotypic and physiological changes under stresses induced by salt, alkali, and drought. Furthermore, overexpression of LpNAC6 resulted in enhanced stress tolerance as evidenced by increased levels of SOD, POD, CAT enzymes, improved photosynthetic indices, and elevated chlorophyll contents; as well as reduced levels of MDA and reactive oxygen species (ROS). These findings demonstrate that we have successfully established a transgenic transformation method for L. pumilum while also providing essential information for cultivating stress-tolerant Lilium species and advancing our understanding of the functions of LpNAC6 in plants.

Improving transient gene expression and agroinfiltration‐based transformation effectiveness in Indonesian orchid Phalaenopsis amabilis (L.) Blume

Transient gene expression is an approach used to study transient genes across various species, with infiltration by Agrobacterium tumefaciens (agroinfiltration) being a commonly used method. Agroinfiltration offers a simple and effective means of delivering transgenes into the plant genome. An alternative method for enhancing the quality and productivity of orchids as ornamental plants is genetic modification through agroinfiltration. Although Agrobacterium‐mediated genetic transformation by immersion has been used on the Phalaenopsis amabilis (L.) Blume species of orchid, transformation efficiency using the immersion technique remains relatively low and the method itself is challenging due to its requirement for aseptic handling. The application of agroinfiltration in P. amabilis has not previously been reported. This study investigates the impact of the injection site, acetosyringone concentration, bacterial density (OD600), and injection volume to determine the optimum conditions for agroinfiltration on P. amabilis. The results demonstrated that injection site had a noticeably distinct impact on transformation effectiveness, with the abaxial position of the leaf being the optimal site for Agrobacterium culture suspension injection. While adjustments in acetosyringone concentration, bacterial density (OD600), and injection volume did not significantly affect transformation efficiency, they did influence the peak time of GFP fluorescence. Acetosyringone at a concentration of 200 µM, an OD600 of 1.0 for Agrobacterium culture, and an injection volume of 500 µL effectively accelerated GFP expression duration.

Refinement and Enhancement of Agrobacterium-Mediated Transient Transformation for Functional Gene Examination in Mulberry (Morus L.)

Background: Mulberry (Morus L.), a vital perennial woody plant with significant economic importance, is utilized for silkworm rearing, human consumption and medicinal use. The availability of mulberry’s whole-genome sequencing data has underscored the demand for an effective, user-friendly, and high-throughput protocol to facilitate the elucidation of gene functions. Methods and Results: In this investigation, we established a transient transformation approach using Agrobacterium tumefaciens-mediated sonication followed by vacuum infiltration in mulberry tissue culture seedlings. Simultaneously, we optimized the transformation conditions, including mulberry genotypes, A. tumefaciens strain, acetosyringone concentration, bacterial density, sonication time, and days after agroinfiltration. These optimizations aimed to achieve heightened transformation efficiency, employing GFP as a reporter gene to monitor transformation events. The optimized method included the use of an infiltration medium (10 mM MgCl2, 10 mM MES (2-(N-morpholino)ethanesulfonic acid sodium salt), 150 μM acetosyringone, and OD600 0.5 of A. tumefaciens LBA4404) supplemented with the surfactant 0.02% Silwet L-77, with 20 s sonication followed by 20 min vacuum infiltration (0.07 MPa). Among the four mulberry genotypes, ‘Taiguo’ was the most responsive genotype and produced the highest levels of GFP expression at 7 d after infiltration. Furthermore, the optimized transient transformation approach has been proven to be successfully applicable for transiently overexpressing MaANS and MaDFR in mulberry fruits of ‘Taiguo’, in vitro, which distinctly enhanced fruit coloring and significantly increased anthocyanin accumulation, respectively. Conclusions: In summary, we devised a dependable, stable and highly efficient transient transformation approach suitable for rapid gene function examination in mulberry leaves and fruits, in vitro.

Construction and optimization of the TRV-mediated VIGS system in Areca catechu embryoids

Areca catechu, a member of the palm family Arecaceae native to tropical regions, holds significant economic importance as a tropical crop. This study explores the application of virus-induced gene silencing (VIGS) in A. catechu embryogenic callus tissues, utilizing the phytoene desaturase gene (AcPDS) of A. catechu as a reporter gene. Results demonstrate that employing EHA105 strain, an Agrobacterium infection liquid concentration of OD600 = 0.5, an infection duration of 5 min, acetosyringone (AS) concentration of 21.5 mg/L, co-cultivation at 19 °C for 2 days followed by 28 °C for 3 days, and a subsequent 14-day post-co-cultivation period, the experimental group (pTRV1+pTRV2-AcPDS) exhibited significantly reduced gene expression compared to the control group CK (pTRV1+pTRV2), manifesting the anticipated photobleaching phenotype by the 21st day. RT-PCR analysis revealed that AcPDS gene expression in TRV-mediated photobleached A. catechu embryos decreased to 0.227 times that of the control group CK. This established VIGS system in A. catechu embryoids establishes an effective experimental platform for studying functional genes in A. catechu.

Tobacco Plant: A Novel and Promising Heterologous Bioreactor for the Production of Recombinant Bovine Chymosin.

The natural source of chymosin, a key enzyme in the dairy industry, is insufficient for rapidly growing cheese industries. Large-scale production of recombinant proteins in heterologous hosts provides an efficient alternative solution. Here, the codon-optimized synthetic prochymosin gene, which has a CAI index of 0.926, was subcloned from a cloning vector (pUC57-bCYM) into the pBI121 vector, resulting in the construct named pBI121-bCYM. CAI ranges from 0 to 1 and higher CAI improves gene expression in heterologous hosts. The overexpression of the prochymosin gene was under the control of constitutive CaMV 35S promoter and NOS terminator and was transferred into the tobacco via A. tumefaciens strain LBA4404. Explant type, regeneration method, inoculation temperature, cell density (OD600) of Agrobacterium for inoculation, and acetosyringone concentration were leaf explants, direct somatic embryogenesis, 19 °C, 0.1, and 100 µM, respectively. The successful integration and expression of the prochymosin gene, along with the bioactivity of recombinant chymosin, were confirmed by PCR, RT-PCR, and milk coagulation assay, respectively. Overall, this study reports the first successful overexpression of the codon-optimized prochymosin form of the bovine chymosin enzyme in the tobacco via indirect transformation. Production of recombinant bovine chymosin in plants can be an easy-to-scale-up, safe, and inexpensive platform.

Establishment of a Rapid and Effective Agrobacterium-Mediated Genetic Transformation System of Oxalis triangularis 'Purpurea'.

Oxalis triangularis 'Purpurea' has significant ornamental value in landscaping. There is a critical necessity to elucidate the gene functions of O. triangularis 'Purpurea' and dissect the molecular mechanisms governing key ornamental traits. However, a reliable genetic transformation method remains elusive. In this study, our investigation revealed that various transformation parameters, including recipient material (petioles), pre-culture time (2-5 days), acetosyringone (AS) concentration (100-400 μM), Agrobacterium concentrations (OD600 = 0.4-1.0), infection time (5-20 min), and co-culture time (2-5 days), significantly impacted the stable genetic transformation in O. triangular 'Purpurea'. Notably, the highest genetic transformation rate was achieved from the leaf discs pre-cultured for 3 days, treated with 200 μM AS infected with Agrobacterium for 11 min at OD600 of 0.6, and subsequently co-cultured for 3 days. This treatment resulted in a genetic transformation efficiency of 9.88%, and it only took 79 days to produce transgenic plants. Our transformation protocol offers advantages of speed, efficiency, and simplicity, which will greatly facilitate genetic transformation for O. triangular 'Purpurea' and gene function studies.

Establishment of Agrobacterium rhizogenes-mediated RNAi of Phryma leptostachya accelerates the functional identification of key genes of the furofuran lignan biosynthetic pathway

Phryma leptostachya is a well-known medicinal plant that produces furofuran lignans with various biological activities. However, the biosynthetic pathways of these natural products in P. leptostachya remain unclear. Hairy root cultures provide a novel method for elucidating the biosynthetic pathways of secondary metabolites. Here, we established a hairy root system for P. leptostachya by optimizing five parameters: Agrobacterium strains, explant types, infection time, A. rhizogenes cell density, and acetosyringone (AS) concentration. The highest transformation frequency (74.51 %) was achieved when the stem explants were infected with ATCC15834 (OD600 = 0.8) strain of A. rhizogenes for 30 min on 1/2 MS medium supplemented with 200 µM AS. Based on the optimal protocol, RNAi hairy roots were induced by silencing the cytochrome P450 gene, PlCYP81Q38, which converts (+)-pinoresinol to (+)-sesamin by means of (+)-piperitol in P. leptostachya, to determine its in vivo role in the lignan biosynthetic pathway and verify the availability of this system. PlCYP81Q38 silencing resulted in significant down-regulation of the PlCYP81Q38 gene at the transcriptional level, consistent with a remarkable decrease in the content of leptostachyol acetate and 6-desmethoxy-leptostachyol acetate, demonstrating its significance in the production of lignans. In conclusion, the approaches and results presented here provide a solid foundation for elucidating the role of key genes involved in the lignan biosynthesis pathway in P. leptostachya and for the industrial production of lignan through plant biotechnology.

Study on induction of Allium sativum L. hairy roots with antibacterial activity against Vibrio

Allium sativum L. has long been used to improve the health of humans and other species due to its antibacterial activity. Applying biotechnology to create a stable and large-scale production process for Allium sativum L., medicinal herbs by hairy root culture technology are essential. However, Allium sativum L. is a monocotyledonous plant, so its ability to induce hairy roots is lower than that of dicotyledonous plants, which requires more research investment. In this study, we succeeded in inducing hairy roots in monocotyledonous plants Allium sativum L, by evaluating parameters impacting hairy root induction such as plant organs, acetosyringone concentration, infection time and co-culture time. The results showed that the optimal procedure for induction of Allium sativum L. hairy roots by Agrobacterium rhizogenes ATCC 15834 was as follows: 10 day old in vitro Allium sativum L. sprouts were wound and soaked in 100 μM acetosyringone solution for 10 min, infection time was 5 minutes, co-culture time was 48 hours cultured on B5 medium (Gamborg B5 medium) having the induction rate of hairy roots reaching 64.4% after 14 days of culture. Allium sativum L. hairy roots had good antibacterial effect against all three strains of Vibrio cholerae, Vibrio parahaemolyticus and Vibrio vulnificus because the MBC/MIC ratios are all less than or equal to 2. Especially, for V. cholerae (MIC = 1.84 mg/ml, MBC = 2.11 mg/ml), hairy roots have antibacterial activity equivalent to tetracycline antibiotics and better than non-infectious roots.

Styrax japonicus functional genomics: An efficient virus induced gene silencing (VIGS) system

VIGS (Virus-induced gene silencing), a method for posttranscriptional gene silencing, is an effective technique for investigating the activities of genes in plants. Since there is no report for available VIGS system in Styrax japonicus, the application of a VIGS approach that results in a gene knockdown to study gene function is limited. In this study, we compared the characteristics that could affect the viability of VIGS in S. japonicus, including the acetosyringone (AS) concentration, the Agrobacterium's optical density and the inoculation method. The stable reference genes of S. japonicus were selected to validate the gene's knockdown by quantitative PCR. As a result, we successfully constructed 2 VIGS systems based on TRV virus: vacuum with AS concentration of 200 μmol·L−1 and OD600 of 0.5, and friction-osmosis with AS concentration of 200 μmol·L−1 and OD600 of 1.0, which silencing efficiency was 83.33% and 74.19%, respectively. The successfully applied VIGS method provides a rapid and effective reverse gene functional analysis approach in S. japonicus to identify unknown gene functions.

The establishment of transient expression systems and their application for gene function analysis of flavonoid biosynthesis in Carthamus tinctorius L

BackgroundSafflower (Carthamus tinctorius L.) is an important economic crop and a traditional medicinal material rich in flavonoids, which can alleviate cardiovascular and cerebrovascular pathologies. Thus, many candidate genes involved in safflower flavonoid biosynthesis have been cloned. However, owing to the lack of a homologous gene expression system, research on gene function is limited to model plants. Therefore, a gene function identification protocol for safflower must be established.ResultsIn the present study, using safflower callus as the experimental material, Agrobacterium and biolistic transient expression systems were established. In the Agrobacterium transient expression system, the highest transformation rate was obtained at the original Agrobacterium concentration of OD600 0.4, infiltration concentration of OD600 0.6, infection for 20 min, co-culture for 3 days, and acetosyringone concentration of 100 μmol·L−1. In the biolistic transient expression system, the highest transformation efficiency was observed at helium pressure of 1,350 psi, vacuum degree of -0.8 bar, flight distance of 6.5 cm, one round of bombardment, plasmid concentration of 3 μg·shot−1, and gold particle concentration of 100 μg·shot−1. Further, these two transient expression systems were used for the functional analysis of CtCHS1 as an example. After overexpression, relative CtCHS1 expression increased, particularly in Agrobacterium-transformed calli. Additionally, the contents of some flavonoids were altered; for instance, naringenin and genistein levels were significantly increased in Agrobacterium-transformed calli, whereas luteolin, luteolin-7-O-rutinoside, and apigenin derivative levels were significantly decreased in biolistic-transformed calli.ConclusionUsing safflower callus as the experimental material, highly efficient Agrobacterium and biolistic transient expression systems were successfully established, and the utility of both systems for investigating gene function was demonstrated. The proposed safflower callus transient expression systems will be useful for further functional analyses of flavonoid biosynthetic genes in safflower.

Establishment of a stable grape immature zygotic embryo-based genetic transformation system

Grapevine, along with other woody perennials, has proven to be a recalcitrant crop to produce transgenic plants, largely restricting its potential for further breed improvement. Here we report the establishment of a stable and reproducible transformation protocol using immature zygotic embryos from 'Chardonnay' seeds. We extensively investigated parameters considered essential for a successful transformation, including antibiotic concentration, pretreatment method, Agrobacterium concentration, concentration of acetosyringone (AS), duration of immature zygotic embryo infection, and the infection method. Our results indicated that transformable immature zygotic embryos may be generated by soaking seeds in a 2.5 g·L−1 gibberellin (GA3) solution for 15 min in a 55 °C constant temperature water bath, followed by 24 h of growth at room temperature before sterilized and inoculated. Furthermore, the transformation rate increased to 12.38% when the immature zygotic embryos of split beak were immersed in the Agrobacterium tumefaciens solution containing 200 μM AS (OD600 = 0.6) for 20 min after 5 days of pre-culture. Transgene integration and expression was further confirmed by the detection of GFP and Western blot in leaves. The newly-developed method took roughly 4 months to obtain the transformation-positive plants, which marked a considerable reduction in the length of time required for the procedure. More importantly, the receptor material is easily accessible, the process unaffected by the changing of the season, and it is straightforward to implement. Our developed transformation protocol on grape immature zygotic embryos offers a tool for gene function analysis and germplasm enhancement via biotechnology.

Highly efficient transient gene expression of three tissues in Osmanthus fragrans mediated by Agrobacterium tumefaciens

As one of the 10 most famous flowers, sweet osmanthus (Osmanthus fragrans) is widely utilized in landscaping and food processing in China. Owing to the high complexity and large size of its genome, it is still an enormous challenge to develop transgenic technology for O. fragrans. Therefore, it is necessary to develop an efficient transient expression protocol for the rapid identification of gene functions in O. fragrans. In this study, we selected three different tissues of O. fragrans, including calli, floral buds, and leaves, as explants for transient transformation mediated by Agrobacterium tumefaciens. However, we found that one protocol did not work for all tissues. Calli were induced and proliferated from the immature plumule of O. fragrans on one-half Murashige and Skoog media that contained 0.5 mg·L−1 6-benzylaminopurine and 1 mg·L−1 naphthalene acetic acid. GUS (β-glucuronidase) was used as a reporter gene, where its level of transcription reflected the abundance of Agrobacterium-mediated T-DNA transferred into explants. To obtain the optimal transient expression protocol for each explant, we evaluated the effects of Agrobacterium load, acetosyringone concentration, co-culture duration, bud bract removal, and explant preculture on the transformation efficiency. The use of our transient expression protocols to overexpress a transcriptional repressor of OfXTH14, OfMYB4, in calli, floral buds, and leaves showed that the relative levels of expression of OfXTH14 decreased significantly in all three of our selected tissues. Since the transformed explants exhibited a transient high level of expression of the target gene, affecting the transcription of related genes as well as the synthesis of related metabolites, they can be widely applied to identify gene functions and study the molecular regulatory mechanisms of O. fragrans. Moreover, the critical steps to significantly increase the transformation efficiency varied from tissue to tissue, providing a reference for the development of transient expression protocols in other plant species.

Agrobacterium rhizogenes mediated genetic transformation in Ficus religiosa L. and optimization of acetylcholinesterase inhibitory activity in hairy roots

The use of decoctions prepared from stem bark to treat neurodegenerative problems is amongst the widespread applications of Ficus religiosa L., a tree of great ethno-medicinal importance in the Indian sub-continent. The stem bark of the tree has been reported to have significant acetylcholinesterase inhibitory activity (AChEI). Latter is one of the most effective modalities used for the treatment of Alzheimer's disease (AD). The development of any commercial treatment modality for AD from this tree faces serious challenges for sample consistency in terms of quality (being strongly influenced by environmental and developmental stages) and quantity. Hairy root (HR) cultures are an excellent choice for such studies. Since no study has been reported for HR culture systems for F. religiosa , the present study was targeted to develop one. Three different HR lines were induced and cultured on different media. Root extracts, obtained from HRs on different media, were subjected to an AChEI assay to select the best conditions where HRs exerted higher AChEI. The wild-type strain of A. rhizogenes was used to induce hairy roots in different explants. The method of genetic transformation was optimised by changing concentrations of bacterial suspension, acetosyringone and calcium chloride in the infection and co-cultivation medium. The assay for measuring AChEI activity was modified from the assays described by Ellman et al. (1961) and Ingkaninan et al. (2000) . These methods recommend the use of IC 50 (half maximal inhibitory concentration) value for comparing AChEI activities in different samples. We developed a protocol for induction and long-term maintenance of HR cultures of F. religiosa L. In vitro conditions were optimized where hairy root extracts exerted higher AChEI activity (IC 50 value of 0.16 µg/ml) compared to that in the stem segments of the mother plants (IC 50 value of 0.49 µg/ml). The present work has been granted a patent by the Indian Patent Office, Government of India (Patent No360050, Date of grant 02/03/2021) (Siwach and Gill, 2021). The present study confirms the utility of F. religiosa in AD treatment and provides a standardized protocol for large-scale production of HR cultures (75ml medium in Erlenmeyer flask of 250 ml volume) that have higher AChEI activity; these cultures offer excellent platforms for future characterization and commercial preparation.

An efficient Agrobacterium-mediated transient transformation system and its application in gene function elucidation in Paeonia lactiflora Pall.

Paeonia lactiflora Pall. is known as the king of herbaceous flowers with high ornamental and precious medicinal value. However, the lack of a stable genetic transformation system has greatly affected the research of gene function in P. lactiflora. The Agrobacterium-mediated transient gene expression is a powerful tool for the characterization of gene function in plants. In this study, the seedlings of P. lactiflora were used as the transformation receptor materials, and the efficient transient transformation system with a GUS reporter gene was successfully established by Agrobacterium harboring pCAMBIA1301. To optimize the system, we investigated the effects of germination time, Agrobacterium cell density, infection time, acetosyringone (AS) concentration, co-culture time, negative pressure intensity, Tween-20 concentration and different receptor materials on the transient transformation efficiency of P. lactiflora. The results showed that the highest transient transformation efficiency (93.3%) could be obtained when seedlings in 2-3 cm bud length were subjected to 12 h infection of resuspension solution comprising 1.2 OD600 Agrobacterium, 200 μM AS and 0.01% Tween-20 under 10 of negative pressure intensity followed by 3 days of co-culture in darkness condition. This method is more suitable for the study of gene function in P. lactiflora. Subsequently, stress resistance genes PlGPAT, PlDHN2 and PlHD-Zip were used to verify the effectiveness of this transformation system. These results can provide critical information for identification of key genes in non-model plants, such as P. lactiflora, and promote the development of molecular biology research for P. lactiflora.

P351 Genetic recombination between the main human skin commensal fungus, Malassezia restricta, and M. globosa using an Agrobacterium tumefaciens-mediated gene transfer system

Poster session 3, September 23, 2022, 12:30 PM - 1:30 PMObjects: Malassezia restricta and M. globosa cause or exacerbate Malassezia -associated skin, seborrheic, and atopic dermatitis, as well as pityriasis versicolor, but the virulence factors remain unclear because between-species genetic recombination has not yet been achieved. We built an Agrobacterium tumefaciens-mediated gene transfer (ATMT) system that generated gene-knockout mutants of both Malassezia species.Materials and Methods: The binary vector pAg1-Δfkb1::NAT1 was introduced into M. restricta CBS 7877 and M. globosa CBS 7966 via ATMT; FKB1 was replaced. Then, FKB1 was re-introduced into the mutants to counteract the deficiencies.Results and Discussion: The medium acetosyringone concentration and temperature, as well as the co-cultivation ratios of A. tumefaciens and the Malassezia strains, affected recombination efficacy. We generated a mutant of the FKB1 gene (which encodes the FKBP12 protein that binds to the calcineurin inhibitor tacrolimus). Wild-type M. restricta and M. globosa were tacrolimus-sensitive, while the FKB1 mutants were tacrolimus-resistant; drug susceptibility was restored by reintroducing FKB1.ConclusionStudies on recombination between M. restricta and M. globosa will aid elucidation of the molecular mechanisms underlying Malassezia -associated dermatitis.

Exploring the Agrobacterium-mediated transformation with CRISPR/Cas9 in cucumber (Cucumis sativus L.).

The narrow genetic basis of cucumber makes breeding of this species difficult. CRISPR/Cas9 system is characteristicof simpledesign, low cost and high efficiency, whichhas opened a new path for cucumber functional genetics and the development of cucumber mocular breeding. However, the immature genetic transformation system is the main limiting factor for applying this technology in cucumber. In this study, a Histochemical β-glucuronidase (GUS) assay was used to analyze the effect of various parameters, including slight scratch of explants, pre-culture time, acetosyringone (AS) concentration, infection time in Agrobacterium solution, and co-culture period on the transformation efficiency. The results showed that the explants slightly scratched after cutting, pre-cultured for 1day, Agrobacterium bacterial solution containing AS, and 20min length of infection could significantly increase the GUS staining rate of explants. On this basis, two sequences with high specificity (sgRNA-1 and sgRNA-2) targeted different loci of gene CsGCN5 were designed. The corresponding vectors Cas9-sgRNA-1 and Cas9-sgRNA-2 were constructed and transformed using the above-optimized cucumber genetic transformation system, and three and two PCR positive lines were obtained from 210 and 207 explants, respectively. No sequence mutation at target loci of CsGCN5 was detected in the Cas9-sgRNA-1 transformed three PCR positive lines. However, one mutant line with targeted homozygous change was recognized from the Cas9-sgRNA-2 transformed two PCR positive lines. In this study, 2.4‰ of total explants had directed mutation in the CsGCN5 gene. The results in the present study would be beneficial to furtheroptimize and improve the efficiency of the genetic transformation of cucumber.

An Attempt to Establish an Agrobacterium-mediated Transient Expression in Euphorbia tirucalli (L.) an Important Medicinal Plant

Background: Optimization of co-cultivation parameters during Agrobacterium-mediated transformation to Euphorbia tirucalli evaluated were bacterial density, infection period, acetosyringone (AS) concentration and co-cultivation temperature. Optimized parameters resulted in high transformation efficiency 3 fold increase at transient GUS expression .The optimized conditions were Agrobacterium tumefaciens growth phase of A600nm 0.17, infection period of 30 min, addition of acetosyringone (AS) in co-cultivation medium (100 µM) and cocultivation temperature of 25°C. Methods: Nodal explants were used for transformation. Bacterial culture was added to 50 ml of liquid YEP medium with kanamycin and rifampicin and grown until reaching the growth phase (A600nm). Bacterial density ranged from A600nm 0.17, 0.56, 0.8 and 1.2 OD were used in the present study. The co-cultivation medium made of solid MS medium consisted of NAA 2 mg L-1 and various concentrations of AS at 0, 50, 100, 200, 400, 600 and 800 µM. Histochemical analysis of gus gene expression was carried out. Result: Higher bacterial density resulted in more transformation efficiency, but also higher necrosis in the explants. Dilution of bacterial suspension reduced necrosis in explants and resulted in higher transformation. The transformation efficiency is 72% when the infection process was carried out with acetosyringone in co-cultivation medium (100 µM). Our studies proved that among the optimized conditions, cocultivation temperature and acetosyringone were the critical parameters during Agrobacterium mediated transformation.

Optimization of Agrobacterium tumefaciens-mediated Transformation in Oxalis corniculata (L.)

Background: Optimization of co-cultivation parameters during Agrobacterium-mediated transformation to Oxalis corniculata evaluated were bacterial density, infection period, acetosyringone (AS) concentration and co-cultivation temperature. Five fold transformation efficiency is achieved in transient gus gene expression after optimizing various parameters. The optimized conditions were Agrobacterium tumefaciens growth phase of A600nm 0.17, infection period of 30 min, addition of acetosyringone (AS) in co-cultivation medium (400 μM) and cocultivation temperature of 22°C. Methods: Oxalis corniculata (L.) were used for transformation. Bacterial culture was added to 50 ml of liquid YEP medium with kanamycin and rifampicin and grown until reaching the growth phase (A600nm). Bacterial density ranged from A600nm 0.17, 0.56,0.80 and 1.34 OD were used in the present study. The co-cultivation medium made of solid MS medium consisted of BAP 2 mg L-1 and NAA 0.5 mg L-1 and various concentrations of AS at 0, 50, 100, 200, 400,600 and 800 μM. Histochemical analysis of gene expression was carried out. Result: Higher bacterial density resulted in more transformation efficiency, but also higher necrosis in the explants. Dilution of bacterial suspension reduced necrosis in explants and resulted in higher transformation. The transformation efficiency is 64% when the infection process was carried out with acetosyringone in co-cultivation medium (400 μM). Our studies proved that among the optimized conditions, cocultivation temperature and acetosyringone were the critical parameters during Agrobacterium mediated transformation.

Highly efficient Agrobacterium-mediated transformation and plant regeneration system for genome engineering in tomato

Tomato (Solanum lycopersicum L.) is an important vegetable and nutritious crop plant worldwide. They are rich sources of several indispensable compounds such as lycopene, minerals, vitamins, carotenoids, essential amino acids, and bioactive polyphenols. Plant regeneration and Agrobacterium-mediated genetic transformation system from different explants in various genotypes of tomato are necessary for genetic improvement. Among diverse plant growth regulator (PGR) combinations and concentrations tested, Zeatin (ZEA) at 2.0 mg l−1 in combination with 0.1 mg l−1 indole-3-acetic acid (IAA) generated the most shoots/explant from the cotyledon of Arka Vikas (36.48 shoots/explant) and PED (24.68 shoots/explant), respectively. The hypocotyl explant produced 28.76 shoots/explant in Arka Vikas and 19.44 shoots/explant in PED. In contrast, leaf explant induced 23.54 shoots/explant in Arka Vikas and 17.64 shoots/explant in PED. The obtained multiple shoot buds from three explant types were elongated on a medium fortified with Gibberellic acid (GA3) (1.0 mg l−1), IAA (0.5 mg l−1), and ZEA (0.5 mg l−1) in both the cultivars. The rooting was observed on a medium amended with 0.5 mg l−1 indole 3-butyric acid (IBA). The transformation efficiency was significantly improved by optimizing the pre-culture of explants, co-cultivation duration, bacterial density and infection time, and acetosyringone concentration. The presence of transgenes in the plant genome was validated using different methods like histochemical GUS assay, Polymerase Chain Reaction (PCR), and Southern blotting. The transformation efficiency was 42.8% in PED and 64.6% in Arka Vikas. A highly repeatable plant regeneration protocol was established by manipulating various plant growth regulators (PGRs) in two tomato cultivars (Arka Vikas and PED). The Agrobacterium-mediated transformation method was optimized using different explants like cotyledon, hypocotyl, and leaf of two tomato genotypes. The present study could be favourable to transferring desirable traits and precise genome editing techniques to develop superior tomato genotypes.