Agrobacterium-mediated genetic transformation of Miscanthus sinensis

Abstract

Miscanthus species are tall perennial rhizomatous grasses with C4 photosynthesis originating from East Asia, and they are considered as important bioenergy crops for biomass production. In this study, Agrobacterium-mediated transformation system for M. sinensis was developed using embryogenic calli derived from mature seeds. In order to establish a stable system, optimum conditions to obtain highly regenerable and transformation-competent embryogenic calli were investigated, and embryogenic calli were efficiently induced with callus induction medium containing 3 mg L -1 2,4-dichlorophenoxyacetic acid and 25 mM L-proline, at pH 5.7 with an induction temperature of 28°C. In addition, the embryogenic callus induction and regeneration potentials were compared between seven M. sinensis germplasms collected from several sites in Korea, which revealed that the germplasm SNU-M-045 had superior embryogenic callus induction and regeneration potentials. With this germplasm, the genetic transformation of M. sinensis was performed using Agrobacterium tumefaciens EHA105 carrying pCAM-BIA1300 with a green fluorescence protein gene as a reporter. After putative transgenic plants were obtained, the genomic integration of transgenes was confirmed by genomic PCR, transgene expression was validated by Northern blot analysis, and the number of transgene integration was confirmed by DNA gel blot analysis. Furthermore, the Agrobacteriummediated transformation of M. sinensis was also performed with pCAMBIA3301 which contains an herbicide resistance gene (BAR), and we obtained transgenic M. sinensis plants whose herbicide resistance was confirmed by spraying with BASTA Ò . Therefore, we have established a stable Agrobacterium-mediated transformation system for M. sinensis, and also successfully produced herbicide-resistant Miscanthus plants by introducing BAR gene via the established method.

Agrobacterium tumefaciens-mediated transformation of axillary bud callus of Hibiscus rosa-sinensis L. ‘Ruby’ and regeneration of transgenic plants

“…Procedures to produce transformed plants are usually empirically determined as the type of explant, and the ratio and type of PGR needed for successful transformation and regeneration, can vary considerably depending upon plant genotype. This has clearly been illustrated for kenaf (Herath et al 2005), Prunus avium (Feeney et al 2007), chickpea (Tripathi et al 2013) and Miscanthus (Hwang et al 2014). In Actinidia eriantha for example, BAP in combination with zeatin was critical for successful plant regeneration, including those of transformed plants (Wang et al 2006).…”

Breeding progress and preparedness for mass‐scale deployment of perennial lignocellulosic biomass crops switchgrass, miscanthus, willow and poplar

“…Robust and effective in vitro regeneration systems have been developed for Miscanthus sinensis , M. × giganteus and M. sacchariflorus (Dalton, ; Guo et al, ; Hwang, Cho, et al, ; Rambaud et al, ; Ślusarkiewicz‐Jarzina et al, ; Wang et al, ; Zhang et al, ). However, there is still significant genotype specificity and these methods need “in‐house” optimization and development to be used routinely.…”

“…They provide potential routes for rapid clonal propagation and also as a basis for genetic transformation. Stable transformation using both biolistics (Wang et al, ) and Agrobacterium tumefaciens DNA delivery methods (Hwang, Cho, et al, ; Hwang, Lim, et al, ) has been achieved in M. sinensis . The development of miscanthus transformation and gene editing to generate diplogametes for producing seed‐propagated triploid hybrids are performed as part of the French project MISEDIT (miscanthus gene editing for seed‐propagated triploids).…”

“…In the United States and United Kingdom, independent and interconnected bi-parental "mapping" families have been studied (Dong et al, 2018;Gifford, Chae, Swaminathan, Moose, & Juvik, 2015) alongside panels of diverse germplasm accessions for GWAS . Further developments calibrating GS with very large panels of parents and cross progeny are underway (Dalton, 2013;Guo et al, 2013;Hwang, Cho, et al, 2014;Rambaud et al, 2013;Ślusarkiewicz-Jarzina et al, 2017;Wang et al, 2011;Zhang et al, 2012). However, there is still significant genotype specificity and these methods need "in-house" optimization and development to be used routinely.…”

Agrobacterium tumefaciens mediated genetic transformation of Ocimum gratissimum: A medicinally important crop

“…The concentration of 300 M was used for further transformation experiments. The various concentrations of AS were used in genetic transformation studies (Pandey et al, 2013;Hwang et al, 2014).…”