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Subject: search.filters.subject.Agrobacterium rhizogenes

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    Functional characterization of genes involved in legume nodulation using hairy root cultures
    (Springer Nature Publishing AG, 2020) Singh, Jawahar; Kumar, Kamal; Verma, Praveen K.
    Legumes, the second most important crop to humans possess unique ability to fix atmospheric nitrogen, making them one of the major contributors to sustainable agriculture. In legumes, molecular characterization of genes by stable transformation is difficult due to their recalcitrant nature to the whole-plant regeneration in desired varieties. The Agrobacterium rhizogenes-mediated generation of transgenic hairy roots or composite plants may facilitate a rapid and convenient alternative to study nodule biology. Functional analysis of genes involved in legume nodulation has been proven as successful for model legumes, viz., Medicago truncatula and Lotus japonicus, using transgenic hairy roots. Besides sharing some common features of nodulation among legumes, the symbiotic signaling is a complex and specific process. Here, we describe an improved protocol for hairy root transformation of a legume crop chickpea (Cicer arietinum L.) and the method to study nodulation to uncover the signaling components. Using the described protocol, transgenic hairy roots were generated in chickpea and selected based on the red fluorescence protein (RFP) microscopy. This protocol can be extended to other underutilized legumes.
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    Establishment of Agrobacterium rhizogenes-mediated hairy root transformation of Crocus sativus L
    (Springer Nature Publishing AG, 2021) Sharma, Shilpi; Singh, Yeshveer; Verma, Praveen K.
    Efficient transformation system for genetic improvement is essential in Crocus sativus, as it lacks sexual reproduction. This is the first report wherein an efficient protocol is developed for the transformation of Crocus sativus L. by Agrobacterium rhizogenes strain ARqua1 with a transformation efficiency of 78.51%. The ARqua1 strain harboring both Ri plasmid and binary vector plasmid pSITE-4NB, and marker genes for red fluorescent protein (RFP) and a β-glucuronidase (GUS) reporter gene were used for selection. Transformation was confirmed by RFP signal, GUS reporter assay and polymerase chain reaction (PCR) analysis of the test samples after 21 days post inoculation. These results confirm the establishment of protocol for hairy root transformation in C. sativus that can be further used for gene transfer or gene editing in Crocus for its genetic improvement.
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    Genotype-independent Agrobacterium rhizogenes-mediated root transformation of chickpea: a rapid and efficient method for reverse genetics studies
    (BioMed Central Ltd, 2018) Aggarwal, Pooja Rani; Nag, Papri; Choudhary, Pooja; Chakraborty, Niranjan; Chakraborty, Subhra
    Background: Chickpea (Cicer arietinum L.), an important legume crop is one of the major source of dietary protein. Developing an efcient and reproducible transformation method is imperative to expedite functional genomics studies in this crop. Here, we present an optimized and detailed procedure for Agrobacterium rhizogenes-mediated root transformation of chickpea. Results: Transformation positive roots were obtained on selection medium after two weeks of A. rhizogenes inoculation. Expression of green fuorescent protein further confrmed the success of transformation. We demonstrate that our method adequately transforms chickpea roots at early developmental stage with high efciency. In addition, root transformation was found to be genotype-independent and the efcacy of our protocol was highest in two (Annigiri and JG-62) of the seven tested chickpea genotypes. Next, we present the functional analysis of chickpea hairy roots by expressing Arabidopsis TRANSPARENT TESTA 2 (AtTT2) gene involved in proanthocyanidins biosynthesis. Overexpression of AtTT2 enhanced the level of proanthocyanidins in hairy roots that led to the decreased colonization of fungal pathogen, Fusarium oxysporum. Furthermore, the induction of transgenic roots does not afect functional studies involving infection of roots by fungal pathogen. Conclusions: Transgenic roots expressing genes of interest will be useful in downstream functional characterization using reverse genetics studies. It requires 1 day to perform the root transformation protocol described in this study and the roots expressing transgene can be maintained for 3–4 weeks, providing sufcient time for further functional studies. Overall, the current methodology will greatly facilitate the functional genomics analyses of candidate genes in root-rhizosphere interaction in this recalcitrant but economically important legume crop.
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    Overexpression of an apoplastic peroxidase gene CrPrx in transgenic hairy root lines of Catharanthus roseus
    (Springer Science, 2011) Jaggi, Monika; Kumar, Santosh; Sinha, Alok Krishna
    Peroxidases are a family of isoenzymes found in all higher plants and are known to be involved in a broad range of physiological processes. However, very little information is available concerning their role in Catharanthus roseus. The present study describes the impact of both overexpression and suppression of a peroxidase gene, CrPrx in C. roseus transgenic hairy root lines. Real-time PCR analysis in 35S-CrPrx and CrPrx-RNAi transgenic lines indicated differential transcript profile for peroxidases as well as for genes and regulators involved in MIA (monoterpenoid indole alkaloid) pathway of C. roseus. Comparative analysis revealed that MIA pathway genes showing elevated levels of expression in 35S-CrPrx transgenic lines showed a significant reduction in their transcript level in CrPrx-RNAi transgenic lines. Metabolite analysis detected higher levels of ajmalicine and serpentine accumulation in overexpressed lines. It was observed that all overexpressed transgenic lines produced more amount of H(2)O(2). These results indicate a role of CrPrx gene in the regulation of MIA pathway genes and regulators, thus affecting the production of specific alkaloids.
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    Effect of loss of T-DNA genes on MIA biosynthetic pathway gene regulation and alkaloid accumulation in Catharanthus roseus hairy roots
    (Springer, 2010) Taneja, Jyoti; Jaggi, Monika; Wankhede, Dhammaprakash Pandhari; Sinha, Alok Krishna
    Hairy roots are generated by integration of T-DNA in host plant genome from root inducing (Ri) plasmid of Agrobacterium rhizogenes and have been utilized for production of secondary metabolites in different plant systems. In Catharanthus roseus, hairy roots are known to show different morphologies, growth patterns, and alkaloid contents. It is also known that during transformation, there is a differential loss of a few T-DNA genes. To decipher the effect of loss of T-DNA genes on the various aspects of hairy roots, ten hairy root clones were analyzed for the presence or absence of T-DNA genes and its implications. It was found that the loss of a few ORFs drastically affects the growth and morphological patterns of hairy roots. The absence of T(R)-DNA from hairy roots revealed increased transcript accumulation and higher alkaloid concentrations, whereas callusing among hairy root lines led to decreased transcript and alkaloid accumulation. Significantly higher expression of MIA biosynthetic pathway genes and low abundance of regulator transcripts in hairy root clones in comparison with non-transformed control roots were also observed. This study indicates that it is not only the integration of T-DNA at certain region of host plant genome but also the presence or absence of important ORFs that affects the expression patterns of MIA biosynthetic pathway genes, regulators, and accumulation of specific alkaloids.