Transgenic sugarcane plants via microprojectile bombardment

Abstract

SummaryTransgenic sugarcane plants were produced by bombardment of embryogenic callus with high-velocity DNA-coated microprojectiles, followed by a selection and regeneration procedure designed for this target tissue. Optimal bombardment conditions for embryogenic callus required microprojectile velocities higher than those previously found effective for sugarcane suspension culture cells. Bombardment of target tissues twice increased the number of transiently expressing cells in regenerable callus regions, to more than 300 per treated plate. Stable transformants were obtained following bombardment with the neomycin phosphotransferase (npt-I/) gene under the control of the Emu strong monocot promoter. Stepped increases in antibiotic concentration during selection and regeneration allowed recovery of actively growing callus and plants on media containing geneticin concentrations completely inhibitory to untransformed controls. NPT-II levels in transformed plants were 20-60 times the background levels in control plants in ELISA assays, and Southern analysis revealed integration of one to three copies of the introduced gene in the sugarcane genome. The procedures described yield one to three transgenic plants per treated plate within 16 weeks of bombardment and provide a simple, efficient and broadly applicable system for genetic transformation of sugarcane. A similar approach should be applicable to other members of the Poaceae able to form embryogenic callus.

Morfogênese in vitro e susceptibilidade de calos de variedades nacionais de cana-de-açúcar (Saccharum officinarum L.) a agentes seletivos utilizados em sistemas de transformação genética

“…A expressão transiente de genes exógenos em protoplastos e a expressão estável em calos foram descritas por , utilizando eletroporação. Plantas transgênicas foram obtidas através da utilização de métodos diretos e indiretos de transformação, incluindo a eletroporação de tecidos meristemáticos (Arencibia et al 1992) e fragmentos de calo (Arencibia et al 1995), o bombardeamento de calos embriogênicos (Bower & Birch 1992, Gallo-Meagher & Irvine 1996 e outras técnicas baseadas na utilização da Agrobacterium tumefaciens (Arencibia et al 1997, Enríquez-Obrigón et al 1998.…”

Genetic transformation of the C4 plant, Flaveria bidentis

“…Although transformation of maize (Fromm eta/., 1990; Gordon-Kamm eta/., 1990; Rhodes et al, 1988) and sugarcane (Bower and Birch, 1992) has been reported, neither system is efficient enough to support the analysis of a large number of gene constructions over a reasonably short time period.…”

An improved rice transformation system using the biolistic method

“…In the past few years, the biolistic method has emerged LS a simple and promising alternative for cereal transformation, and has been successfully used in maize, rice, sugarcane and whoat transformation (Fromm et al 1990; Gordon-Kamm et uf. Christou et al 1991;Bower and Birch, 1992;Vasil et al 1992). Procedures using organized tissues such as immature embryos (Christou et al 1991) or embryo slices (Cao et al 1991) as target tissues for rice transformation may make the system less genotype-dependent.…”