Gene Transfer Into Intact Sugarcane Cells Using Microprojectile Bombardment

Abstract

A microprojectile accelerator has been constructed and used to bombard cultured sugarcane tissues with GUS reporter gene constructs. Design features useful to minimise target tissue damage and variation between shots are described. Transient expression of GUS occurred in pEmuGN-bombarded cells of nonregenerable suspension culture as well as in regenerable embryogenic callus of commercial sugarcane cultivar 463, and in suspension cultures capable of regeneration to plants. Parameters yielding transient GUS expression in up to 1055 cells per bombardment in homogeneous suspension cultures of sugarcane have been established with a mean of 206 expressing cells per bombardment over a series of 8 independent experiments. Approximately 4% of these transiently expressing cells continued to express GUS for extended periods, indicating probable stable transformation of intact cells of the commercial sugarcane cultivar. Microprojectile bombardment appears the most promising of the available gene transfer techniques for practical genetic transformation of sugarcane because most commercial cultivars readily form regenerable callus suitable for bombardment.

Genetic Engineering of Saccharum

“…The impact force from high velocity propelled microparticles breaks the plant cell wall and plasma membrane and allows the introduction of the DNA into or adjacent to the nucleus. This technique is very simple and applicable to a diverse range of regenerable tissues targeted for transformation Franks and Birch 1991 ;Lakshmanan et al 2005 ) . Early studies in the development and optimization of sugarcane transformation identi fi ed the target tissue and the method for selection and regeneration of transformants as the most important factors for the production of stably transformed plants by particle bombardment .…”

“…Since the generation of the fi rst transgenic sugarcane plants from particle bombarded embryogenic callus as target tissue (Bower and Birch 1992 ) , protocols for particle bombardment-mediated transformation of sugarcane relied on the use of embryogenic callus (Bower and Birch 1992 ;Bower et al 1996 ;Snyman 2004 ) , even though meristematic tissues (Gambley et al 1993(Gambley et al , 1994 and suspension cells (Chowdhury and Vasil 1992 ;Franks and Birch 1991 ) were successfully transformed. More recently, young leaf roll disks with or without preemergent in fl orescence (also called immature in fl orescence) have emerged as a suitable target for transformation (Snyman et al 2000(Snyman et al , 2006 .…”

Genetic Transformation in Buffel Grass (Cenchrus ciliaris L.)

“…Maximal velocity, obtained by accelerating the macroprojectile from the top of the barrel in the device described by Franks and Birch (1991), generated the highest transient expression levels.…”

“…Pieces of embryogenic callus approximately 0.5 cm in diameter were arranged over a 3-cm diameter filter paper moistened with MS medium containing 3% sucrose, 5% coconut water, and 4 mg/l 2,4-D (MSC4). Plasmid DNA was precipitated onto tungsten particles and accelerated in the device described by Franks and Birch (1991). Bombardments.…”

Transformation of Sugarcane

“…Franks and Birch (1991) established conditions yielding 10 2 to 10 3 transiently expressing cells per bombardment of cells from homogeneous, nonregenerable suspension cultures of cultivar Q63. Approximately 2 to 4% of transiently expressing cells appeared to be stably transformed, as indicated by continued expression of the introduced reporter gene.…”