Genetic Transformation of Recalcitrant Upland Switchgrass Using Morphogenic Genes

Abstract

Switchgrass (Panicum virgatum) is an excellent feedstock for biofuel production. While genetic transformation is routinely done in lowland switchgrass, upland cultivars remain recalcitrant to genetic transformation. Here we report the establishment of an efficient and reproducible transformation protocol for two upland cultivars, ‘Summer’ and ‘Blackwell’, by ectopic overexpression of morphogenic genes, Baby boom (Bbm) and Wuschel2 (Wus2). Two auxotrophic Agrobacterium strains, LBA4404Thy- and EHA105Thy-, each harboring the same construct containing ZmBbm, ZmWus2, and a green fluorescence protein (GFP) gene, ZsGreen1, were used to infect immature leaf segments derived from in vitro grown seedlings. The Agrobacterium strains also contain a transformation helper plasmid that carry additional copies of Agrobacterium virulence genes. GFP-expressing calli were identified and selected for regeneration. The highest transformation efficiency of 6% was obtained for the tetraploid cultivar Summer when LBA4404Thy- was used for infection, which is twice of that for the octoploid cultivar Blackwell. LBA4404Thy- consistently outperformed EHA105Thy- on transformation frequency across the two cultivars. Fifteen randomly selected putative transgenic plants of Summer and Blackwell, representing independent callus events, were confirmed as transgenic by the presence of the transgene, ZmAls, and the absence of AtuFtsZ, a chromosomal gene specific to the Agrobacterium strain LBA4404 using polymerase chain reaction. Transgene integration and expression was further confirmed by the detection of GFP in roots, and the resistance to herbicide injury to leaves of selected putative transgenic plants. The ZmBbm and ZmWus2 genes were successfully removed from 40 to 33.3% of the transgenic plants of Summer and Blackwell, respectively, via the Cre-Lox recombination system upon heat treatment of GFP-expressing embryogenic calli. Our successful transformation of recalcitrant upland switchgrass provides a method for gene function analysis and germplasm enhancement via biotechnology.

Leaf transformation for efficient random integration and targeted genome modification in maize and sorghum

“…The use of Wus2 and Bbm expanded the list of alternative explants for transformation, as demonstrated by the regeneration of fertile transgenic plants using mature embryo slices and leaf-base tissue segments in maize 10 . Using constructs developed by Lowe et al 10 , recent reports have shown leaf-base transformation in switchgrass 27 and successful gene mutagenesis in Eragrostis tef 28 . However, further improvements are needed to increase transformation frequency, to expand the method within the grass family and to eventually enable more complex genome editing applications.…”

“…Lowe et al 10 have shown that the use of morphogenic genes Wus2 and Bbm facilitated direct Agrobacterium-mediated transformation of basal leaf tissue and the recovery of maize plants with random transgene integration. Recently, this method has been extended to random transformation in Panicum virgatum 27 and Cas9-mediated mutagenesis in Eragrostis tef 28 , demonstrating that Wus2/Bbm can facilitate direct leaf-base transformation in other grass species. However, to support the true potential of Cas9-mediated genome editing, substantially higher transformation rates are still required to produce the large numbers of plants necessary to recover complex genome modifications, such as targeted HDR-mediated gene insertion and large-scale chromosomal rearrangements 9 .…”

Tissue culture and Agrobacterium-mediated genetic transformation of the oil crop sunflower

“…Genetic transformation is usually carried by Agrobacterium -mediated method, its transformation efficiency is often influenced by different factors, such as Agrobacterium strains, bacterial density, duration of infection, co-culture time, acetosyringone concentration, and the type of antibiotics [ 20 ], and so on. The gene was successfully transformed into Hevea brasiliensis using cotyledonary somatic embryos as explants [ 21 ], similarly, immature leaf segments of Switchgrass ( Panicum virgatum ) [ 22 ] and leaves of dandelion ( Taraxacum officinale ) were successfully transformed to obtain positive seedlings by optimizing various conditions [ 23 ]. Genetic transformation in sunflower is less reported.…”

Foxtail mosaic virus-induced gene silencing (VIGS) in switchgrass (Panicum virgatum L.)

“…However, the genetic diversity and high self-incompatibility of switchgrass ecotypes have limited genetic studies and the development of mutant resources [ 6 , 7 ]. Transformation protocols and methods for CRISPR/Cas9- and RNAi-mediated gene editing in switchgrass have been reported [ 8 – 13 ]. For example, the pANIC vector collection has been successfully employed for the stable downregulation of lignin biosynthetic genes in transgenic embryogenic callus cultures [ 14 , 15 ] and have been used in transgenic RNAi studies to analyze and regulate cellulose and lignin biosynthesis in switchgrass [ 10 , 16 ].…”