Embryogenic cell suspensions for high-capacity genetic transformation and regeneration of switchgrass (Panicum virgatum L.)

Christine A Ondzighi-Assoume,Wayne A Parrott,Sara M Allen,C Neal Stewart,Jason N Burris,Jonathan D Willis,Wilson K Ouma,Scott C Lenaghan,Wusheng Liu,Zachary King

doi:10.1186/s13068-019-1632-3

Abstract

BackgroundSwitchgrass (Panicum virgatum L.), a North American prairie grassland species, is a potential lignocellulosic biofuel feedstock owing to its wide adaptability and biomass production. Production and genetic manipulation of switchgrass should be useful to improve its biomass composition and production for bioenergy applications. The goal of this project was to develop a high-throughput stable switchgrass transformation method using Agrobacterium tumefaciens with subsequent plant regeneration.ResultsRegenerable embryogenic cell suspension cultures were established from friable type II callus-derived inflorescences using two genotypes selected from the synthetic switchgrass variety ‘Performer’ tissue culture lines 32 and 605. The cell suspension cultures were composed of a heterogeneous fine mixture culture of single cells and aggregates. Agrobacterium tumefaciens strain GV3101 was optimum to transfer into cells the pANIC-10A vector with a hygromycin-selectable marker gene and a pporRFP orange fluorescent protein marker gene at an 85% transformation efficiency. Liquid cultures gave rise to embryogenic callus and then shoots, of which up to 94% formed roots. The resulting transgenic plants were phenotypically indistinguishable from the non-transgenic parent lines.ConclusionThe new cell suspension-based protocol enables high-throughput Agrobacterium-mediated transformation and regeneration of switchgrass in which plants are recovered within 6–7 months from culture establishment.

Highlights

Switchgrass (Panicum virgatum L.), a North American prairie grassland species, is a potential lignocellu‐ losic biofuel feedstock owing to its wide adaptability and biomass production
Using the fresh weight (FW) of cells as a parameter, we found that Panicum virgatum var. Performer32 (P32) and Panicum virgatum var. Performer605 (P605) cell suspension cultures displayed an increase of the density of cells over 14 days of the culture, reaching the growth phase by 6 days of culture (Fig. 1i)
Using the loss of weight by dissimilation (LWD) [40] of P32 and P605 cell suspension cultures over 14 days, we found that this dissimilation was elevated over the time (Additional file 1: Figure S1), indicating that these two cell lines underwent cell division and/or enlargement

Summary

Introduction

Switchgrass (Panicum virgatum L.), a North American prairie grassland species, is a potential lignocellu‐ losic biofuel feedstock owing to its wide adaptability and biomass production. Production and genetic manipulation of switchgrass should be useful to improve its biomass composition and production for bioenergy applications. The goal of this project was to develop a high-throughput stable switchgrass transformation method using Agrobacterium tumefaciens with subsequent plant regeneration. Switchgrass is considered to be recalcitrant for genetic transformation and depends on the ability of explants (cells or tissues) to regenerate whole plants in culture. Switchgrass is a good candidate species for biotechnological innovations vis-à-vis cell biology and genetics

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