Hairy Root Transformation in Lotus japonicus

Okamoto, Satoru; Yoro, Emiko; Suzaki, Takuya; Kawaguchi, Masayoshi

doi:10.21769/bioprotoc.795

Cited by 14 publications

(11 citation statements)

References 3 publications

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“…In the present work we studied the two Ljnbcl1:LORE1 insertion mutants (lines 30119830 and 30053558; Figure a) and performed RNA interference (RNAi) approaches targeting LjNBCL1 transcripts ( Ljnbcl1 :RNAi; Figure a) via hairy‐root transformation (Kumagai and Kouchi, ; Okamoto et al ., ). The impact of Ljnbcl1:LORE1 insertion (line 30053558) and Ljnbcl1 :RNAi on LjNBCL1 transcript accumulation was assessed by quantitative (q)RT‐PCR in 35‐dpi nodules.…”

Section: Resultsmentioning

confidence: 97%

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Lotus japonicus NOOT‐BOP‐COCH‐LIKE1 is essential for nodule, nectary, leaf and flower development

et al. 2018

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The NOOT-BOP-COCH-LIKE (NBCL) genes are orthologs of Arabidopsis thaliana BLADE-ON-PETIOLE1/2. The NBCLs are developmental regulators essential for plant shaping, mainly through the regulation of organ boundaries, the promotion of lateral organ differentiation and the acquisition of organ identity. In addition to their roles in leaf, stipule and flower development, NBCLs are required for maintaining the identity of indeterminate nitrogen-fixing nodules with persistent meristems in legumes. In legumes forming determinate nodules, without persistent meristem, the roles of NBCL genes are not known. We thus investigated the role of Lotus japonicus NOOT-BOP-COCH-LIKE1 (LjNBCL1) in determinate nodule identity and studied its functions in aerial organ development using LORE1 insertional mutants and RNA interference-mediated silencing approaches. In Lotus, LjNBCL1 is involved in leaf patterning and participates in the regulation of axillary outgrowth. Wild-type Lotus leaves are composed of five leaflets and possess a pair of nectaries at the leaf axil. Legumes such as pea and Medicago have a pair of stipules, rather than nectaries, at the base of their leaves. In Ljnbcl1, nectary development is abolished, demonstrating that nectaries and stipules share a common evolutionary origin. In addition, ectopic roots arising from nodule vascular meristems and reorganization of the nodule vascular bundle vessels were observed on Ljnbcl1 nodules. This demonstrates that NBCL functions are conserved in both indeterminate and determinate nodules through the maintenance of nodule vascular bundle identity. In contrast to its role in floral patterning described in other plants, LjNBCL1 appears essential for the development of both secondary inflorescence meristem and floral meristem.

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Section: Resultsmentioning

confidence: 97%

“…Lotus japonicus hairy‐root transformations were performed as described in Kumagai and Kouchi () and Okamoto et al . () with minor modifications using the A. rhizogenes ARqua1 strain. Hairy roots were subsequently inoculated with M. loti and nodules were observed at 35 dpi.…”

Section: Methodsmentioning

confidence: 99%

Lotus japonicus NOOT‐BOP‐COCH‐LIKE1 is essential for nodule, nectary, leaf and flower development

et al. 2018

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“…Hairy root transformation was conducted according to Okamoto et al (2013). In short, hypocotyls of L. japonicus seedlings were dipped in A. rhizogenes AR1193 culture transformed with a plasmid carrying an mCherry marker driven by a UBIQUITIN promoter (called EV in Pimprikar et al, 2016).…”

Section: Hairy Root Transformationmentioning

confidence: 99%

“…Therefore, hairy root transformation is a commonly used technique in root symbiosis research on model legumes to generate transgenic roots for experimental use (e.g., to study promoter activity with reporter constructs, or to express tagged proteins) in a shorter period of time (Boisson-Dernièr et al, 2001). After dipping seedling hypocotyls into A. rhizogenes solution it takes 2 weeks until hairy roots of 2-5 cm develop (Okamoto et al, 2013). Depending on the purpose of the experiment or the plant genotype, the time to grow hairy roots with a usable size can take even longer.…”

Section: Growth Of Lotus Japonicus Hairy Roots In Hydroponic Culturementioning

confidence: 99%

A Flexible, Low-Cost Hydroponic Co-Cultivation System for Studying Arbuscular Mycorrhiza Symbiosis

et al. 2020

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Arbuscular mycorrhiza (AM) is a widespread symbiosis between plant roots and fungi of the Glomeromycotina, which improves nutrient uptake by plants. The molecular mechanisms underlying development and function of the symbiosis are subject to increasing research activity. Since AM occurs in the soil, most studies targeting a molecular understanding of AM development and function, use solid substrates for cocultivating plants and AM fungi. However, for some experiments very clean roots, highly controlled nutrient conditions or applications of defined concentrations of signaling molecules (such as hormones) or other small chemicals (such as synthetic inhibitors or signaling agonists) are needed. To this end, hydroponics has been widely used in research on mechanisms of plant nutrition and some hydroponic systems were developed for AM fungal spore amplification. Here, we present a hydroponics setup , which can be successfully utilized for experimental root colonization assays. We established a "tipwick" system based on pipette tips and rock wool wicks for co-cultivation of AM fungi with small model plants such as Lotus japonicus. A larger "Falcon-wick" system using Falcon tubes and rockwool wicks was developed for larger model plants such as rice. The hydroponic system can also be employed for growing L. japonicus hairy roots after transformation by Agrobacterium rhizogenes, thus circumventing the laborious cultivation on agar medium-containing Petri dishes during hairy root development. The tip-wick and Falcon-wick systems are easy to use and can be built with low cost, conventional and reusable lab plastic ware and a simple aquarium pump.

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“…Therefore, it is important to establish a transient expression system in L. japonicus for rapid evaluation of gene function, such as sub-cellular protein localization. Studies on L. japonicus transformation have predominantly used Agrobacterium rhizogenes-mediated hairy root transformation where genes of interest can be transiently expressed specifically in the roots (Okamoto et al 2013). Other transient gene expression systems such as those using protoplasts (Jia et al 2018) and calli (Kimura et al 2015) have been reported.…”

mentioning

confidence: 99%

Agroinfiltration-based efficient transient protein expression in leguminous plants

Suzaki

Tsuda

Ezura

et al. 2019

Plant Biotechnology

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Transient protein expression is an effective tool to rapidly unravel novel gene functions, such as transcriptional activity of promoters and sub-cellular localization of proteins. However, transient expression is not applicable to some species and varieties because of insufficient expression levels. We recently developed one of the strongest agroinfiltrationbased transient protein expression systems for plant cells, termed 'Tsukuba system. ' About 4 mg/g fresh weight of protein expression in Nicotiana benthamiana was obtained using this system. The vector pBYR2HS, which contains a geminiviral replication system and a double terminator, can be used in various plant species and varieties, including lettuces, eggplants, tomatoes, hot peppers, and orchids. In this study, we assessed the applicability of the Tsukuba system to several species of legumes, including Lotus japonicus, soybean Glycine max, and common bean Phaseolus vulgaris. The GFP protein was transiently expressed in the seedpods of all examined legume species, however, protein expression in leaves was observed only in P. vulgaris. Taken together, our system is an effective tool to examine gene function rapidly in several legume species based on transient protein expression.

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Hairy Root Transformation in Lotus japonicus

Cited by 14 publications

References 3 publications

Lotus japonicus NOOT‐BOP‐COCH‐LIKE1 is essential for nodule, nectary, leaf and flower development

Lotus japonicus NOOT‐BOP‐COCH‐LIKE1 is essential for nodule, nectary, leaf and flower development

A Flexible, Low-Cost Hydroponic Co-Cultivation System for Studying Arbuscular Mycorrhiza Symbiosis

Agroinfiltration-based efficient transient protein expression in leguminous plants

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