A simple and reliable multi-gene transformation method for switchgrass

Abstract

A simple and reliable Agrobacterium -mediated transformation method was developed for switchgrass. Using this method, many transgenic plants carrying multiple genes-of-interest could be produced without untransformed escape. Switchgrass (Panicum virgatum L.) is a promising biomass crop for bioenergy. To obtain transgenic switchgrass plants carrying a multi-gene trait in a simple manner, an Agrobacterium-mediated transformation method was established by constructing a Gateway-based binary vector, optimizing transformation conditions and developing a novel selection method. A MultiRound Gateway-compatible destination binary vector carrying the bar selectable marker gene, pHKGB110, was constructed to introduce multiple genes of interest in a single transformation. Two reporter gene expression cassettes, GUSPlus and gfp, were constructed independently on two entry vectors and then introduced into a single T-DNA region of pHKGB110 via sequential LR reactions. Agrobacterium tumefaciens EHA101 carrying the resultant binary vector pHKGB112 and caryopsis-derived compact embryogenic calli were used for transformation experiments. Prolonged cocultivation for 7 days followed by cultivation on media containing meropenem improved transformation efficiency without overgrowth of Agrobacterium, which was, however, not inhibited by cefotaxime or Timentin. In addition, untransformed escape shoots were completely eliminated during the rooting stage by direct dipping the putatively transformed shoots into the herbicide Basta solution for a few seconds, designated as the 'herbicide dipping method'. It was also demonstrated that more than 90 % of the bar-positive transformants carried both reporters delivered from pHKGB112. This simple and reliable transformation method, which incorporates a new selection technique and the use of a MultiRound Gateway-based binary vector, would be suitable for producing a large number of transgenic lines carrying multiple genes.

Long-term maintenance of high regeneration ability of switchgrass embryogenic callus

“…In fact, we have successfully obtained transgenic switchgrass plants from 18-monthold calli (Ogawa et al 2014). These findings indicate that caryopsis-derived Type I callus is useful source for genetic transformation of switchgrass.…”

Producing Transgenic Thompson Seedless Grape (Vitis vinifera) Plants using Agrobacterium tumefaciens

“…In this study, 24 explants out of 100 survived on the bialaphos containing selection medium and were successfully transformed grapevine clones. This limited number of successfully transformed grapevine clones is not uncommon and has been attributed by various researchers to several different factor that influence the relative low transformation efficiency including initial difficulties experienced in the introduction of foreign genes and the selection and regeneration of transformed cells in grape species (Perl and Eshdat, 1998), escape genes during selection (Ogawa et al, 2014), problems in the production of roots increasing the time to get plantlets (Luth and Moore, 1999), trouble in conditions required for successful Agrobacterium infection, and also identifying the suitable Agrobacterium strain and selectable marker gene (Carimi et al, 2012). Several methods for genetic transformation have been attempted for grape, but only Agrobacterium mediated transformation has been previously successful (Vivier and Pretorius, 2000).…”

An efficient <i>Agrobacterium</i>-mediated transformation method for switchgrass genotypes using Type I callus

“…Six-to twelve-month-old caryopsis-derived Type I calli were transformed using the Agrobacterium-mediated method of Ogawa et al (2014) with modifications. One callus line of cultivar ' Alamo' , A09-01, was used for optimization of transformation conditions.…”

“…We previously found that the transformation efficiency of caryopsis-derived Type I calli could be increased by prolonged cocultivation (Ogawa et al 2014). In the present study, we aimed to establish a simple and highly efficient Agrobacterium-mediated transformation method using caryopsis-derived Type I calli of switchgrass by focusing on the cocultivation conditions as well as the preculture conditions.…”

“…However, switchgrass is a self-incompatible outcrossing species, so all cultivars are genotypically variable. Different genotypes within a cultivar affect the transformation efficiency (Ogawa et al 2014;Somleva et al 2002) and potentially affect characteristics of transgenic plants that carry the same transgene(s). Seedling-derived callus has been induced and used for transformation (Song et al 2012), although these callus lines also comprise different genotypes.…”