A Simple and Efficient Protocol for Hairy Root Culture of Arabidopsis Thaliana

Hairy root culture (HRC) is a valuable biotechnological tool for the production of plant secondary metabolites. Secondary metabolome study of Arabidopsis thaliana can be helpful in understanding the biological roles of various secondary metabolites present in it and HRC constructs the base of such study. The present work deals with the establishment of Agrobacterium rhizogenes strains transformed HRC of A. thaliana with very high transformation frequency resulting in long term hairy root cultures grown in hormone free media. Optimization of culture medium and standardisation of co-cultivation period are the key role players in obtaining high frequency of hairy roots. Four days of preculture in CIM medium and five min of co-cultivation in the bacterial suspension were found to be optimal conditions for root induction. This protocol could become a powerful tool for transcriptomics and proteomics-based studies for different transgenic root lines of A. thaliana.

Preparation of electrocompetent cells v2

This protocol is used to prepare electrocompetent cells with high transformation efficiency.

Preparation of electrocompetent cells v2

This protocol is used to prepare electrocompetent cells with high transformation efficiency.

Preparation of electrocompetent cells v2

This protocol is used to prepare electrocompetent cells with high transformation efficiency.

Preparation of electrocompetent cells v1

This protocol is used to prepare electrocompetent cells with high transformation efficiency.

On the Decrease in Transformation Stress in a Bicrystal Cu-Al-Mn Shape-Memory Alloy during Cyclic Compressive Deformation

The evolution of the inhomogeneous distribution of the transformation stress (σs) and strain fields with an increasing number of cycles in two differently orientated grains is investigated for the first time using a combined technique of digital image correlation and data-driven identification. The theoretical transformation strains (εT) of these two grains with crystal orientations [5 3 26]β and [6 5 11]β along the loading direction are 10.1% and 7.1%, respectively. The grain with lower εT has a higher σs initially and a faster decrease in σs compared with the grain with higher εT. The results show that the grains with higher σs might trigger more dislocations during the martensite transformation, and thus result in greater residual strain and a larger decrease in σs during subsequent cycles. Grain boundary kinking in bicrystal induces an additional decrease in transformation stress. We conclude that a grain with crystal orientation that has high transformation strain and low transformation stress (with respect to loading direction) will exhibit stable transformation stress, and thus lead to higher functional performance in Cu-based shape memory alloys.

Exploration of an Efficient Electroporation System for Heterologous Gene Expression in the Genome of Methanotroph

One-carbon (C1) substrates such as methane and methanol have been considered as the next-generation carbon source in industrial biotechnology with the characteristics of low cost, availability, and bioconvertibility. Recently, methanotrophic bacteria naturally capable of converting C1 substrates have drawn attractive attention for their promising applications in C1-based biomanufacturing for the production of chemicals or fuels. Although genetic tools have been explored for metabolically engineered methanotroph construction, there is still a lack of efficient methods for heterologous gene expression in methanotrophs. Here, a rapid and efficient electroporation method with a high transformation efficiency was developed for a robust methanotroph of Methylomicrobium buryatense 5GB1. Based on the homologous recombination and high transformation efficiency, gene deletion and heterologous gene expression can be simultaneously achieved by direct electroporation of PCR-generated linear DNA fragments. In this study, the influence of several key parameters (competent cell preparation, electroporation condition, recovery time, and antibiotic concentration) on the transformation efficiency was investigated for optimum conditions. The maximum electroporation efficiency of 719 ± 22.5 CFU/μg DNA was reached, which presents a 10-fold improvement. By employing this method, an engineered M. buryatense 5GB1 was constructed to biosynthesize isobutyraldehyde by replacing an endogenous fadE gene in the genome with a heterologous kivd gene. This study provides a potential and efficient strategy and method to facilitate the cell factory construction of methanotrophs.

Agrobacterium-Mediated Transformation of pMDC140 Plasmid Containing the Wheatwin2 Gene into the Tadong Rice Genome

Blast disease resulting from Magnaporthe oryzae fungal infection reduces annual rice yield by up to 30% globally. The wheatwin2 (wwin2) is a pathogenesis-related (PR) gene that encodes for a PR-4 protein with chitinase properties that is capable of degrading chitin, a major constituent of certain fungal cell walls. However, the potential for wwin2 to contribute to M. oryzae resistance in rice is unclear. This study reports the construction of a pMDC140 vector carrying the wwin2 gene and its Agrobacterium-mediated transformation into the Tadong rice genome. In brief, the wwin2 gene was synthesized and integrated into a pMDC140 vector using Gateway cloning technology and was transformed into the Tadong rice genome. Our results show a promising high transformation rate, with more than 90% of the transformed rice calli expressing β-glucuronidase (GUS), the reporter gene marker. The expression of the wwin2 gene in transformed rice calli was further confirmed using quantitative real-time polymerase chain reaction. In conclusion, a pMDC140-wwin2 vector was constructed, which had a high transformation rate and could consistently induce expression of the GUS and wwin2 genes in Tadong rice. Data of this study is beneficial for subsequent in vitro and M. oryzae-infected field experiments to confirm the defense mechanism of the wwin2 gene towards blast disease in rice.

Anti-inflammatory Compounds Produced in Hairy Roots Culture of Sphaeralcea Angustifolia

Sphaeralcea angustifolia (Cav) G. Don is used in traditional Mexican medicine to treat inflammations and gastric disease. Its anti-inflammatory and immunomodulatory activities in mice and rats acute and chronic models have been attributed mainly to scopoletin. Scopoletin reduced joint inflammation, the number of new vessels, production of endogenous angiogenic inducers, and reversed the histopathological alterations in rat adjuvant-induced arthritis. Tomentin and sphaeralcic acid from S. angustifolia cells in suspension proved anti-inflammatory and immunomodulatory activities in mice kaolin/λ-carrageenan-induced arthritis. Transformed roots of S. angustifolia have been proposed as active compounds producers. A high transformation frequency mediated by Agrobacterium rhizogenes ATCC15834/pTDT was obtained from nodal segments (59.5 ± 10.5%, 145 hairy root lines) and leaves (40.0 ± 25, 52 hairy root lines) of 2-month-old plantlets. Among seven lines selected according to their phenotypic characteristics and growth index, the SaTR N7.2 line presented the highest sphaeralcic acid production (17.6 ± 1.72 mg/g DW); this production was 440-fold superior to that reported in S. angustifolia wild plants, and in comparison to cells in the suspension of S. angustifolia in MS medium with nitrate restriction this was 263-fold higher when cultured in flasks and 5-fold higher in a stirred-tank type bioreactor. The SaTR N7.2, SaTR N5.1, SaTR N7.1, and SaTR N15.1 lines excreted sphaeralcic acid into the culture medium at similar levels. Genetic transformation of hairy roots was confirmed by amplifying a 490 bp fragment of the rolC gene. S. angustifolia hairy root cultures producers of scopoletin and sphaeralcic acid can be stressed by nitrate reduction and/or copper increased to stimulate scopoletin and sphaeralcic acid production.