Arlene R. Howe

Rapid and reproducible Agrobacterium-mediated transformation of sorghum

A rapid and reproducible Agrobacterium-mediated transformation protocol for sorghum has been developed. The protocol uses the nptII selectable marker gene with either of the aminoglycosides geneticin or paromomycin. A screen of various A. tumefaciens strains revealed that a novel C58 nopaline chromosomal background carrying the chrysanthopine disarmed Ti plasmid pTiKPSF2, designated NTL4/Chry5, was most efficient for gene transfer to sorghum immature embryos. A NTL4/Chry5 transconjugant harboring the pPTN290 binary plasmid, which carries nptII and GUSPlusTM expression cassettes, was used in a series of stable transformation experiments with Tx430 and C2-97 sorghum genotypes and approximately 80% of these transformation experiments resulted in the recovery of at least one transgenic event. The transformation frequencies among the successful experiments ranged from 0.3 to 4.5%, with the average transformation frequency being approximately 1% for both genotypes. Over 97% of the transgenic events were successfully established in the greenhouse and were fully fertile. Co-expression of GUSPlusTM occurred in 89% of the transgenic T0 events. Seed set for the primary transgenic plants ranged from 145 to 1400 seed/plant. Analysis of T1 progeny demonstrated transmission of the transgenes in a simple Mendelian fashion in the majority of events.

Glyphosate as a selective agent for the production of fertile transgenic maize (Zea mays L.) plants

Efficient and reproducible selection of transgenic cells is an essential component of a good transformation system. In this paper, we describe the development of glyphosate as a selective agent for the recovery of transgenic embryogenic corn callus and the production of plants tolerant to Roundup® herbicide. Glyphosate, the active ingredient in Roundup® herbicide inhibits the enzyme 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) and thus prevents the synthesis of chorismate-derived aromatic amino acids and secondary metabolites in plants. A maize EPSPS gene has been cloned, mutated to produce a modified enzyme resistant to inhibition by glyphosate, and engineered into a monocot expression vector. In addition, a bacterial gene which degrades glyphosate (glyphosate oxidoreductase, or GOX) was also cloned into a similar expression vector. Stably transformed callus has been reproducibly recovered following introduction of mutant maize EPSPS and GOX genes into tissue culture cells by particle bombardment and selection on glyphosate-containing medium. Plants have been regenerated both on and off glyphosate selection medium, and are tolerant to normally lethal levels of Roundup®. Excellent seed set has been obtained from both self and outcross pollinations from both sprayed and unsprayed regenerated plants. Progeny tests have demonstrated normal Mendelian transmission and tolerance to the herbicide for some of the transgenic events.

Sorghum Transformation: Overview and Utility

Over the past decade genomics resources available for sorghum have rapidly expanded (Paterson Int J Plant Genomics 2008:6, 2008), these resources, coupled with the recent completion of the genome sequence which is relatively small in size (730 Mb) (Paterson et al. Nature 457:551–556, 2009) makes sorghum a rather attractive species to study. Moreover, the USDA germplasm system maintains 42,614 accessions, of which more than 800 exotic landraces have been converted to day length-insensitive lines to facilitate their use in breeding programs. In addition, a set of EMS mutation stocks developed by the USDA Plant Stress and Germplasm Development Unit in Lubbock, TX (Xin et al. Bioenerg Res 2:10–16, 2009) will be a valuable resource for functional genomics studies in sorghum. However, in order to be a robust system for study a suite of functional genomics tools are necessary to complement these other resources to aid in down-stream hypothesis testing. A key functional genomics tool is the ability to modulate gene expression through the introduction of transgenic genetic elements. This is exemplified by recent work (Cook et al. Plant Cell 22:867–887, 2010) in which RNAi experiments were employed to specifically reduced expression of two alkylresorcinol synthases to demonstrate their role in the synthesis of the allelopathic molecule sorgoleone. In addition to its value as a functional genomics tool, plant transformation offers a route to broaden access to novel input and output traits for sorghum breeding programs.