Co-cultivation Duration Research Articles

Optimization of regeneration and transformation system in finger millet (Eleucine coracana L.)

A proficient in vitro regeneration protocol and transformation system is a requisite for genetic improvement of finger millet. Finger millet (Eleucine coracana L.) GN-4 was used for optimization of regeneration protocol. Using seed as a explants, highest frequency of callus induction was observed on MS supplemented with 2,4-D (1.0 mg.L-1), Proline (500 mg.L-1) and Casein enzyme hydrolysate (300 mg.L-1). Among the different combinations used for shoot initiation, MS with kinetin (1.50 mg.L-1) and BAP (0.50 mg.L-1) produced maximum number of shoots (11.00) having highest shoot length (11.2 cm). Within six days, maximum numbers of roots (12.3) were obtained on half strength MS basal media fortified with NAA (1 mg.L-1). In hardening process, maximum survival (44.51%) of plantlets with minimum days for new sprouting (4.4 days) was reported in vermicompost: sand: cocopeat (1:1:1 v.v-1) mixture. Agrobacterium tumefaciens mediated genetic transformation system was optimized and putative transgenic plants were confirmed by PCR. When the effect of the age of the callus and co-cultivation duration were evaluated, forty five days old co-cultivated callus for five days yielded 1.33% frequency of transformation.

Agrobacterium-Mediated Genetic Transformation of the Medicinal Plant Veratrum dahuricum.

Veratrum dahuricum L. (Liliaceae), a monocotyledonous species distributed throughout the Changbai mountains of Northeast China, is pharmaceutically important, due to the capacity to produce the anticancer drug cyclopamine. An efficient transformation system of Veratrum dahuricum mediated with Agrobacterium tumefaciens is presented. Murashige and Skoog (MS) medium containing 8 mg/L picloram was used to induce embryogenic calli from immature embryos with 56% efficiency. A. tumefaciens LBA4404 carrying the bar gene driven by the cauliflower mosaic virus 35S promoter was employed for embryogenic callus inoculation. A. tumefaciens cell density OD660 = 0.8 for inoculation, half an hour infection period, and three days of co-culture duration were found to be optimal for callus transformation. Phosphinothricin (PPT, 16 mg/L) was used as the selectable agent, and a transformation efficiency of 15% (transgenic plants/100 infected calli) was obtained. The transgenic nature of the regenerated plants was confirmed by PCR and Southern blot analysis, and expression of the bar gene was detected by RT-PCR and Quick PAT/bar strips. The steroid alkaloids cyclopamine, jervine, and veratramine were detected in transgenic plants, in non-transformed and control plants collected from natural sites. The transformation system constitutes a prerequisite for the production of the pharmaceutically important anticancer drug cyclopamine by metabolic engineering of Veratrum.

An optimized Agrobacterium tumefaciens-mediated transformation system for random insertional mutagenesis in Fonsecaea monophora

Chromoblastomycosis (CBM) is a chronic cutaneous or subcutaneous mycosis that is prevalent worldwide. Though CBM tends not to be fatal, it is difficult to treat and complications can include chronic, marked lesions, lymphatic damage, and neoplastic transformation. Fonsecaea monophora, as a new species segregated from Fonsecaea pedrosoi, is the predominant causative pathogen of CBM in southern China. However, research about F. monophora has been limited, which may be due to a lack of an effective genetic manipulation system for F. monophora. In this study, we successfully established a random insertional mutagenesis system by Agrobacterium tumefaciens-mediated transformation (ATMT) in F. monophora for the first time. In order to improve the efficiency of ATMT, various co-culture conditions were optimized, including: acetosyringone (AS) concentrations, co-culture duration, ratio of bacteria to conidia, and the A. tumefaciens strains. In addition, thermal asymmetric interlaced polymerase chain reaction (TAIL-PCR) was performed to identify the transferred DNA (T-DNA) flanking sequences of the F. monophora transformants. The valuable transformants obtained in this study will be used for research in the future.

Studying the Effects of Tumor-Secreted Paracrine Ligands on Macrophage Activation using Co-Culture with Permeable Membrane Supports.

Tumor-derived paracrine signaling is an overlooked component of local immunosuppression and can lead to a permissive environment for continued cancer growth and metastasis. Paracrine signals can involve cell-cell contact between different cell types, such as PD-L1 expressed on the surface of tumors interacting directly with PD-1 on the surface of T cells, or the secretion of ligands by a tumor cell to affect an immune cell. Here we describe a co-culture method to interrogate the effects of tumor-secreted ligands on immune cell (macrophage) activation. This straightforward procedure utilizes commercially available 0.4 µm polycarbonate membrane permeable supports and standard tissue culture plates. In the process described, macrophages are cultured in the upper chamber and tumor cells in the lower chamber. The presence of the 0.4 µm barrier allows for the study of intercellular signaling without the confounding variable of physical contact, because the two cell types share the same medium and exposure to paracrine ligands. This approach can be combined with others, such as genetic alterations of the macrophage (e.g., isolation from genetic knock-out mice) or tumor (e.g., CRISPR-mediated alterations) to study the role of specific secreted factors and receptors. The approach also lends itself to standard molecular biological analyses such as quantitative reverse transcription polymerase chain reaction (qRT-PCR) or Western blot analysis, without the need for flow sorting to separate the two cell populations. Enzyme-linked immunosorbent assays (ELISAs) can similarly be utilized to measure secreted ligands to better understand the dynamic interaction of cell signaling in the multiple cell type context. Duration of co-culture can also be varied for the study of temporally regulated events. This co-culture method is a robust tool that facilitates the study of tumor-secreted signals in the immune context.

Standardization of Regeneration, Agrobacterium-Mediated Transformation, and Introduction of Nucleocapsid Gene of Watermelon Bud Necrosis Virus in Watermelon

Seven types of explants (proximal cotyledon, distal cotyledon, distal cotyledon leaves, proximal cotyledon leaves, distal hypocotyls, proximal hypocotyls, and basal petioles) of watermelon (Citrullus lanatus) cv. Sugar Baby were tested for their regeneration capacity on MS medium supplemented with different concentrations and combinations of hormones (BAP 0–3 mg/l alone and IAA 0–0.5 mg/l and BAP 0–3 mg/l together). The results showed that the proximal petiole cultured in MS + BAP (3 mg/l) showed the highest percentage of callus induction (28%) and shoot production (24%). The proximal cotyledons cultured in MS + BAP (2 mg/l) + IAA (0.1 mg/l) showed the highest regeneration frequency (76%). Agrobacterium tumefaciens strain EHA 105 carrying a binary vector pBI121 containing the GUS gene (s-glucuronidase) and kanamycin-resistance gene, nptII, was used to transform petiole explants of watermelon cv. Sugar Baby. Various conditions such as the agrobacterial concentration of OD600 0.6, infection time of 20 min, co-cultivation duration of 2 days and selection at 100 mg/l kanamycin were found as important parameters for the successful Agrobacterium-mediated transformation of watermelon. Further, a transgene construct using the nucleocapsid protein (NP) gene from watermelon bud necrosis virus (genus Tospovirus family Peribunyaviridae) was developed in pBI121 and used to transform watermelon. The successful transformation of petiole explant of watermelon with the GUS gene as well as the NP gene was confirmed by molecular assays, which showed a transformation efficiency of 14.2% and 0.375% with the GUS and NP gene, respectively.

Agrobacterium rhizogenes-mediated RNAi of Tripterygium wilfordii and application for functional study of terpenoid biosynthesis pathway genes

Tripterygium wilfordii is an important medical plants which is rich in several valuable bioactive terpenoids, such as triptolide and wilforine. Developing a fast and efficient transformation method is imperative for exploring these metabolites biosynthesis pathway by using RNA interference (RNAi) technology. In the present study, an efficient transformation system using Agrobacterium rhizogenes was established by optimizing four parameters including vacuum infection time, pre-culture duration, appropriate concentration of Agrobacterium, and co-cultivation duration. The transformation efficiency could reach to 62.22% by using leaf sections pre-cultured 5 days as explants, vacuum infection with Agrobacterium at optical density (OD600) of 0.6 for 3 min and co-cultivation for 3 d. The T. wilfordii 3-hydroxy-3-methylglutaryl- coenzyme A reductase (TwHMGR) RNAi hairy root was induced to validate the applicability of this system. The identification of transgenic hairy root was monitored by fluorescence microscopy and verified by PCR analysis. Real-time quantitative PCR analysis showed that the expression of TwHMGR was significantly decreased in transgenic hairy roots compared with control lines. The detection of metabolites showed that the silence of TwHMGR could highly decrease the content of triptolide and wilforine. These results indicated that this A. rhizogenes-mediated RNAi system is an effective protocol for characterizing the function of genes involved in the terpenoid biosynthetic pathway in Tripterygium wilfordii.

In vitro regeneration and Agrobacterium tumefaciens-mediated genetic transformation of D. lotus ( Diospyros lotus L.)

Abstract In vitro regeneration and Agrobacterium-mediated genetic transformation system of D. lotus (Diospyros lotus L.) has been achieved using leaf explants. Two-step regeneration method was developed. First, the explants cultured in callus induction medium containing 0.1 mg/L naphthalene acetic acid (NAA) and 3.0 mg/L thidiazuron (TDZ) and cultured in darkness for 10 days. Second, leaf discs were introduced on shoot induction medium containing 1 mg/L trans-zeatin (ZT) and 0.1 mg/L indole-3-acetic acid (IAA) after 15 days of inoculation, callus induction rate of Diospyros lotus L. was 100%, average number of adventitious shoots was 4.26, regeneration rate was 94.28% respectively. The highest number root formation was observed in MS(1/2 N) supplemented with 0.5 mg/L 6-benzylaminopurine (6-BA) and 1.0 mg/L indole-3-butyric acid (IBA). A protocol for genetic transformation of D. lotus via Agrobacterium was established. Agrobacterium strain EHA105 carrying vector pCAMBIA1301-GA2OX2 was used to optimize the transformation conditions, which include pre-culture period, bacterial optical density, infection time, co-culture duration, concentration of acetosyringone (AS) and concentration of hygromycin. Improved transformation rates (14.27 ± 1.32%) were achieved when explants pre-cultured for 4 days were incubated with an Agrobacterium suspension with a culture density of OD600 = 0.5 for 10 min, followed by 1-day co-cultivation. Successful transformation was confirmed by histochemical GUS activity, hpt and gus gene PCR analyses of the regeneration hygromycin resistant plantlets.

Genotype-independent and enhanced in planta Agrobacterium tumefaciens-mediated genetic transformation of peanut [Arachis hypogaea (L.)

Agrobacterium infection and regeneration of the putatively transformed plant from the explant remains arduous for some crop species like peanut. Henceforth, a competent and reproducible in planta genetic transformation protocol is established for peanut cv. CO7 by standardizing various factors such as pre-culture duration, acetosyringone concentration, duration of co-cultivation, sonication and vacuum infiltration. In the present investigation, Agrobacterium tumefaciens strain EHA105 harboring the binary vector pCAMBIA1301-bar was used for transformation. The two-stage selection was carried out using 4 and 250mgl-1 BASTA® to completely eliminate the chimeric and non-transformed plants. The transgene integration into plant genome was evaluated by GUS histochemical assay, polymerase chain reaction (PCR), and Southern blot hybridization. Among the various combinations and concentrations analyzed, highest transformation efficiency was obtained when the 2-day pre-cultured explants were subjected to sonication for 6min and vacuum infiltrated for 3min in Agrobacterium suspension, and co-cultivated on MS medium supplemented with 150µM acetosyringone for 3days. The fidelity of the standardized in planta transformation method was assessed in five peanut cultivars and all the cultivars responded positively with a transformation efficiency ranging from minimum 31.3% (with cv. CO6) to maximum 38.6% (with cv. TMV7). The in planta transformation method optimized in this study could be beneficial to develop superior peanut cultivars with desirable genetic traits.

Simple, rapid and efficient transformation of genotype Nisqually-1: a basic tool for the first sequenced model tree

Genotype Nisqually-1 is the first model woody plant with an available well-annotated genome. Nevertheless, a simple and rapid transformation of Nisqually-1 remains to be established. Here, we developed a novel shoot regeneration method for Nisqually-1 using leaf petiole and stem segment explants. Numerous shoots formed in the incision of explants within two weeks. The optimized shoot regeneration medium (SRM) contained 0.03 mg l−1 6-benzylaminopurine, 0.02 mg l−1 indole-3-butyric acid and 0.0008 mg l−1 thidiazuron. Based on this, Agrobacterium-mediated genetic transformation of stem explants was examined using the vector pBI121 that contains the β-glucuronidase (GUS) as a reporter gene. Consequently, factors affecting transformation frequency of GUS-positive shoots were optimized as follows: Agrobacteria cell suspension with an OD600 of 0.4, 20 min infection time, 2 days of co-cultivation duration and the addition of 80 µM acetosyringone into Agrobacteria infective suspension and co-cultivation SRM. Using this optimized method, transgenic plantlets of Nisqually-1 – with an average transformation frequency of 26.7% – were obtained with 2 months. Southern blot and GUS activity staining confirmed the integration of the foreign GUS gene into Nisqually-1. This novel transformation system for Nisqually-1 was rapid, efficient, and simple to operate and will improve more genetic applications in this model tree.

Optimization of transient GUS expression of Agrobacterium-mediated transformation in Dierama erectum Hilliard using sonication and Agrobacterium

Efficient Agrobacterium-mediated transformation was established via sonication of embryonic shoot apical meristems (ESAMs) of Dierama erectum Hilliard. Effects of explant types, co-cultivation time, acetosyringone concentration, Agrobacterium concentration and different gene delivery methods were evaluated for higher efficiency of genetic transformation in D. erectum. An explant type (ESAMs), concentration of Agrobacterium inoculum (OD600 of 1.6) and acetosyringone (50mgL−1), and co-cultivation duration (3days) were optimized for efficient genetic transformation of D. erectum. The transformation efficiency varied with explant types (from 0 to 60%), concentrations of bacteria (10 to 55%) and acetosyringone (50 to 90%) and period of co-cultivation (30 to 70%). The transformation efficiency was best with ESAMs explants compared with callus clusters. The gene delivery method via sonication-assisted Agrobacterium-mediated transformation (SAAT) provided higher transformation efficiency (40%) GUS expression compared with agrobacterial monolayer and agrobacterial suspension which gave less than 5% transformation efficiency. The putative transgenic plants which histochemically expressed GUS, were confirmed further with PCR and 35.3% of the plants were GUS positive. Stable integration of the transgene was not demonstrated hence the GUS expression observed was regarded as transient. This newly developed transformation system may facilitate improvement of D. erectum characteristics and other related geophytes.

Ultrastructural investigation of the time-dependent relationship between breast cancer cells and thrombosis induction

Thromboembolic complications are a common cause of death in breast cancer patients. The in vivo relationship between coagulation factors and circulating tumours is a multifaceted one, with tumour cells implicated in thrombocytosis and platelets associated with coagulation-mediated metastasis. Platelets and fibrin networks are known to be morphologically altered in patients with cancer, and their relationship with breast cancer cells themselves may increase thrombosis susceptibility. This was investigated in vitro, assessing such morphological alterations through the establishment of a MCF-7 breast cancer cell co-culture system with blood plasma. Co-culture duration ranged from zero to thirty minutes, with five-minute intervals, in order to assess the time-dependent ultrastructural conformations of platelet and fibrin networks, using scanning electron microscopy. It was found that enhanced coagulability was related to co-culture duration. Changes in platelet and fibrin network morphology from normal were visible as early as five minutes in co-culture with MCF-7 cells. With longer co-culture duration thrombosis-linked variation in structural configuration was intensified, including advanced platelet aggregation and adherence characteristics, compression of fibrin networks into plaques of increased density, and upsurge of microparticulate extrusion implicated in amplifying procoagulant events during the metastatic process. These results confirm that cancer cells are stimulators of coagulation even in an in vitro system and breast cancer patients may become increasingly susceptible to thrombotic-related consequences with increased exposure to tumour cells, especially during metastasis.

An Efficient Protocol for Agrobacterium-mediated Transformation of Sugarcane by Optimizing of Duration of Co-cultivation and Age of Callus

An Efficient Protocol for Agrobacterium-mediated Transformation of Sugarcane by Optimizing of Duration of Co-cultivation and Age of Callus

Induction of hairy roots and over production of anthraquinones in Oldenlandia umbellata L.: a dye yielding medicinal plant by using wild type Agrobacterium rhizogenes strain

Hairy root cultures of Oldenlandia umbellata L. was established by infecting leaf explants with wild type Agrobacterium rhizogenes strain (MTCC 532). Leaf explants were co-cultured with activated bacteria in half strength Murashige and Skoog (MS) basal medium containing 500 mg l−1 ampicillin for the induction of hairy roots. To facilitate effective transfer of T-DNA of Ri plasmid, duration of co-cultivation and concentration of acetosyringone were standardized as 48 h and 200 µM, respectively. Roots were produced from the infected leaves within 14 days. Fast growing roots (2–3 cm) were excised and sub-cultured in half strength basal MS liquid medium for the development of transgenic lines. Thus ten hairy root lines were raised and superior line L3 was selected on the basis of fresh weight (1.5 g), dry weight (0.26 g) and anthraquinone (AQ) content (10.374 mg g−1 dry wt). Superior line (L3) was further cultured to test stability of AQ production and found that stable production of AQ during five consecutive sub-cultures. High performance liquid chromatographic method was adopted for quantification of purpurin from samples. Hairy roots produced 21.83 mg g−1 purpurin, while roots from wild growing plants produced 6.08 mg g−1 purpurin. Thus purpurin content in hairy roots was about 3.6 times higher than wild growing roots. Further, polymerase chain reaction based amplification using primers specific for genes rolA, rolB, and rolC confirmed the transformation.

In vitro regeneration and optimization of factors affecting Agrobacterium mediated transformation in Artemisia Pallens, an important medicinal plant.

Artemisia pallens is an important medicinal plant. In-vitro regeneration and multiplication of A. pallens have been established using attached cotyledons. Different growth regulators were considered for regeneration of multiple shoots. An average of 36 shoots per explants were obtained by culturing attached cotyledons on Murashige and Skoog's medium containing 2mg/L BAP and 0.1mg/L NAA, after 45days. The shoots were rooted best on half Murashige and Skoog's medium with respect to media containing 1mg/L IBA or 1mg/L NAA. Different parameters such as type of bacterial strains, OD600 of bacterial culture, co-cultivation duration, concentration of acetosyringone and explants type were optimized for transient expression of the reporter gene. Agrobacterium tumefaciens harbouring pCambia1301 plasmid carrying β-glucuronidase as a reporter gene and hygromycin phosphotransferase as plant selectable marker genes were used for genetic transformation of A. pallens. Hygromycin lethality test showed concentration of 15mg/L were sufficient to inhibit the growth of attached cotyledons and multiple shoot buds of nontransgenics in selection media. Up to 83% transient transformation was found when attached cotyledons were co-cultivated with Agrobacterium strain AGL1 for 2days at 22°C on shoot induction medium. The bacterial growth was eliminated by addition of cefotaxime (200mg/L) in selection media. T0 transgenic plants were confirmed by GUS histochemical assay and further by polymerase chain reaction (PCR) using uidA and hpt gene specific primers. The study is useful in establishing technological improvement in A. pallens by genetic engineering.

Comprehensive screening of influential factors in the Agrobacterium tumefaciens-mediated transformation of the Himalayan elixir: Ajuga bracteosa Wall. ex. Benth

Ajuga bracteosa is a valuable medicinal plant producing many important compounds, including withanolides, phytoecdysteroids, neo-clerodane-di and triterpenes, iridoid glycosides, flavonoids, etc. These compounds possess a broad spectrum of biological, pharmacological and medicinal properties, but their yield is very low in wild-type plants, and chemical synthesis is not viable. Metabolic engineering strategies offer a promising solution for the bulk production of these natural products. However, these strategies usually require the establishment of an efficient genetic transformation method, which to date has not been reported for A. bracteosa. Therefore, the current study was conducted by employing Agrobacterium tumefaciens C58C1 harboring the binary plasmid p35SGUSint with GUS as the reporter gene and the NTPII gene as the selectable marker. The influential factors, e.g. explant type, optical density of agrobacterial culture, inoculation period, co-cultivation duration and acetosyringone concentration, were systematically investigated and the optimal transformation conditions were established by 72 independent transformation experiments. Putative transformants were selected on kanamycin 100mg/L. MS medium supplemented with 3.6mg/L BA resulted in maximum regeneration frequency of the transformed explants. The expression of the GUS gene was confirmed by histochemistry and polymerase chain reaction. We established that nodal explants of A. bracteosa precultured for 3days, inoculated with a culture of A. tumefaciens with an OD of 1.0 for 20min and co-cultivated for 2days in MS medium with 200μM acetosyringone at media pH 5.8 showed 100% transformation induction. This is the first report of a successful A. tumefaciens-mediated transformation of A. bracteosa.

The effects of co-culture duration of cord blood cells with adipose tissue-derived stromal cells on hematopoietic precursors> amplification

The effects of co-culture duration of cord blood cells with adipose tissue-derived stromal cells on hematopoietic precursors> amplification

Direct shoot organogenesis and Agrobacterium tumefaciens mediated transformation of Solanum trilobatum L.

Solanum trilobatum L. Is one of the most valued medicinal plants of Indian Ayurveda and Siddha medicine. An efficient protocol for direct shoot organogenesis and Agrobacterium tumefaciens mediated transformation using in vitro derived leaf explants of S. Trilobatum was established for the first time. High frequency direct shoot regeneration (89%) was achieved in MS medium containing 2 mg/L 6-benzylaminopurine and 0.25 mg/L indole-3-acetic acid. The Agrobacterium strain EHA 105 harboring the binary vector pCAMBIA 1301, which contained hygromycin phosphotransferase (hpt) as a selectable marker gene and β-glucuronidase (gus) as a reporter gene, was used for the transformation. The factors that influence the transformation frequency were optimized as follows: age of explants (40 days old), acetosyringone concentration (150 μM), duration of preculture (2 days), bacterial optical density (O.D. 0.4), Tween 20 concentration (0.01%), infection time (15 min), pH of cocultivation medium (5.4), and cocultivation duration (3 days). The putative transformants were screened on selection medium containing 15 mg/L hygromycin. The transgenic plants were confirmed by histochemical gus analysis, PCR, and RT-PCR analysis. A stable transformation efficiency of 64% was achieved under optimized transformation conditions. Thus, the protocol can be used for genetic improvement of this plant in the future.

Characterization of the Populus PtrCesA4 promoter in transgenic Populus alba × P. glandulosa

An efficient shoot regeneration method was developed using leaf explants of Populus alba × P. glandulosa, and the optimized shoot regeneration medium contained 0.1 mg L−1 NAA, 0.5 mg L−1 6-BA and 0.002 mg L−1 TDZ. The factors for Agrobacterium-mediated transformation of this hybrid were further optimized as follows: no preculture step, Agrobacterium cell suspension with an OD600 of 0.6, 20 min infection time and 2d co-cultivation duration. To investigate Populus trichocarpa cellulose synthase A4 promoter (PtrCesA4pro), we constructed the PtrCesA4pro:: β-glucuronidase (GUS) binary vector, in which the cauliflower mosaic virus 35S (CaMV35S) promoter of pBI121was replaced with the PtrCesA4pro to drive the expression of a GUS reporter gene. Forty-eight kanamycin-resistant plantlets were obtained through Agrobacterium-mediated transformation of this hybrid. Genomic DNA PCR and Southern blot analyses confirmed the integration of PtrCesA4pro::GUS into this hybrid and the expression of GUS gene was driven by the PtrCesA4 promoter. A histochemical GUS staining presented the activity of PtrCesA4 promoter in the transgenic lines. No GUS signal was observed in leaves, including the veins and petioles, and notably weak staining was detected in the secondary xylems of the roots. The PtrCesA4 promoter was highly active in the fibers, the vessels of developing xylems, and the fibers of mature phloems. Surprisingly, GUS staining was detected in the cambial cells of the highly lignified stems of these transgenic trees. Our results implicate PtrCesA4 promoter as a good genetic tool for controlling gene function in wood development and tree molecular breeding.

Assessment of factors influencing the tissue culture-independent Agrobacterium-mediated in planta genetic transformation of okra [Abelmoschus esculentus (L.) Moench

Regeneration of transformed plants from the Agrobacterium-infected tissue is a time-consuming process and requires hard work. Okra [Abelmoschus esculentus (L.) Moench] is highly recalcitrant to Agrobacterium-mediated genetic transformation and regeneration. In this study, we established a tissue culture-independent genetic transformation system for okra using seed as an explant. Agrobacterium tumefaciens EHA 105 harbouring the binary vector pCAMBIA 1301–bar was used to infect the okra seeds. Various parameters influencing the okra genetic transformation including, co-cultivation duration, acetosyringone, sonication, and vacuum infiltration have been evaluated. Maximum transformation efficiency of 18.3 % was recorded when the pre-cultured okra seeds were sonicated for 30 min and vacuum infiltrated for 3 min in Agrobacterium suspension containing 100 µM acetosyringone and co-cultivated for 3 days on a medium containing 100 µM acetosyringone. The GUS histochemical analysis confirmed the gus A gene integration and expression, whereas polymerase chain reaction (PCR) and Southern blot hybridization confirmed the bar gene integration and copy number in the transformed okra genome. The transgene was successfully segregated into the progeny plants with a Mendelian inheritance ratio of 3:1. The in planta transformation protocol developed in the present investigation is applicable to transform the okra plants with disease-resistant traits, and the transformed plants can be generated within 60 days.

A reliable and high-efficiency Agrobacterium tumefaciens-mediated transformation system of Pogonatherum paniceum embryogenic callus using GFP as a reporter gene

Pogonatherum paniceum (Lam.) Hack, a perennial turfgrass, is important in ecological restoration and landscape construction because of its strong root system and attractive appearance. In this paper, we report the establishment of Agrobacterium tumefaciens-mediated transformation of P. paniceum embryogenic callus (EC) using a high-frequency regeneration system. We also optimised the transformation conditions, such as sub-culture and pre-culture of EC, Agrobacterium concentration, infection time, acetosyringone (AS) concentration, co-cultivation duration, bacteriostatic antibiotics, spectinomycin concentration, washing solutions and selection schemes, based on the transformation efficiency evaluated by green fluorescent protein (GFP) visual analysis, callus survival rate and differentiation rate. The optimal solution was prepared under the following conditions: EC was sub-cultured three times and pre-cultured for 4 days prior to transformation, Agrobacterium concentration of OD600 = 0.6, infection time of 10 min, AS concentration of 40 mg/L, co-cultivation duration of 3 days, washing with liquid washing medium plus 250 mg/L cefotaxime, selection with 300 mg/L cefotaxime and 150 mg/L spectinomycin for 2 or 4 weeks of callus selection, 4 or 2 weeks of differentiation selection and 2 weeks of rooting selection. In the optimised transformation system, the transformation efficiency reached nearly 70 %. PCR and liquid Southern hybridization demonstrated the stable integration of the gfp gene into the P. paniceum genome, GFP visual analysis and RT-PCR confirmed gfp gene expression. This reliable and high-efficiency A. tumefaciens-mediated transformation system lays the foundation for genetic improvement and functional gene research of P. paniceum and improves the application of P. paniceum in ecological restoration and landscape construction.