Plant transformation is now a core research tool in plant biology and a practical tool for cultivar improvement. There are verified methods for stable introduction of novel genes into the nuclear genomes of over 120 diverse plant species. This review examines the criteria to verify plant transformation; the biological and practical requirements for transformation systems; the integration of tissue culture, gene transfer, selection, and transgene expression strategies to achieve transformation in recalcitrant species; and other constraints to plant transformation including regulatory environment, public perceptions, intellectual property, and economics. Because the costs of screening populations showing diverse genetic changes can far exceed the costs of transformation, it is important to distinguish absolute and useful transformation efficiencies. The major technical challenge facing plant transformation biology is the development of methods and constructs to produce a high proportion of plants showing predictable transgene expression without collateral genetic damage. This will require answers to a series of biological and technical questions, some of which are defined.

SummaryCarbon captured through photosynthesis is transported, and sometimes stored in plants, as sugar. All organic compounds in plants trace to carbon from sugars, so sugar metabolism is highly regulated and integrated with development. Sugars stored by plants are important to humans as foods and as renewable feedstocks for industrial conversion to biofuels and biomaterials. For some purposes, sugars have advantages over polymers including starches, cellulose or storage lipids. This review considers progress and prospects in plant metabolic engineering for increased yield of endogenous sugars and for direct production of higher-value sugars and simple sugar derivatives. Opportunities are examined for enhancing export of sugars from leaves. Focus then turns to manipulation of sugar metabolism in sugar-storing sink organs such as fruits, sugarcane culms and sugarbeet tubers. Results from manipulation of suspected 'limiting' enzymes indicate a need for clearer understanding of flux control mechanisms, to achieve enhanced levels of endogenous sugars in crops that are highly selected for this trait. Outcomes from in planta conversion to novel sugars and derivatives range from severe interference with plant development to field demonstration of crops accumulating higher-value sugars at high yields. The differences depend on underlying biological factors including the effects of the novel products on endogenous metabolism, and on biotechnological fine-tuning including developmental expression and compartmentation patterns. Ultimately, osmotic activity may limit the accumulation of sugars to yields below those achievable using polymers; but results indicate the potential for increases above current commercial sugar yields, through metabolic engineering underpinned by improved understanding of plant sugar metabolism.

SummarySucrose is the feedstock for more than half of the world's fuel ethanol production and a major human food. It is harvested primarily from sugarcane and beet. Despite attempts through conventional and molecular breeding, the stored sugar concentration in elite sugarcane cultivars has not been increased for several decades. Recently, genes have been cloned for bacterial isomerase enzymes that convert sucrose into sugars which are not metabolized by plants, but which are digested by humans, with health benefits over sucrose. We hypothesized that an appropriate sucrose isomerase (SI) expression pattern might simultaneously provide a valuable source of beneficial sugars and overcome the sugar yield ceiling in plants. The introduction of an SI gene tailored for vacuolar compartmentation resulted in sugarcane lines with remarkable increases in total stored sugar levels. The high-value sugar isomaltulose was accumulated in storage tissues without any decrease in stored sucrose concentration, resulting in up to doubled total sugar concentrations in harvested juice. The lines with enhanced sugar accumulation also showed increased photosynthesis, sucrose transport and sink strength. This remarkable step above the former ceiling in stored sugar concentration provides a new perspective into plant source-sink relationships, and has substantial potential for enhanced food and biofuel production.

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.