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 pCAMBIA1300 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 Agrobacterium-mediated 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®. 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.
Highlights
The production of biofuel from plant carbohydrates depends on the solar energy stored in plant biomass in thePlant Cell Tiss Organ Cult (2014) 117:51–63 form of soluble sugars, starch and structural polysaccharides through photosynthesis (Gomez et al 2008; Zhu et al 2008)
The important steps for Agrobacterium-mediated transformation of crops include the induction of regenerable embryogenic calli, DNA delivery into the embryogenic calli by the inoculation and co-cultivation of Agrobacterium cells harboring a vector with target gene(s), and the selection of transformed calli and regeneration of transgenic plants (Kim et al 2007; Engler and Jakob 2013)
We used mature seeds-derived embryogenic calli to develop Agrobacterium-mediated transformation system for M. sinensis, as the embryogenic callus tissue derived from mature seeds has been successfully used for Agrobacterium-mediated transformation of several monocotyledons (Cheng et al 2004)
Summary
The production of biofuel from plant carbohydrates depends on the solar energy stored in plant biomass in thePlant Cell Tiss Organ Cult (2014) 117:51–63 form of soluble sugars, starch and structural polysaccharides through photosynthesis (Gomez et al 2008; Zhu et al 2008). Bioethanol has mainly been produced from soluble sugars and starch, since they are more processed into biofuel than structural lignocellulose polysaccharides (Naik et al 2010). This type of production appears unsustainable because of the potential competition with food and feed production. One way to address this problem is to focus on breeding efforts for the development of seed-propagated varieties in genetically stable and fertile species, such as M. sinensis (Christian et al 2005) This is economically attractive, as it will likely lower the cost of planting with a better establishment and speed up the development of Miscanthus as a dedicated biomass crop
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