Effects of tissue culture, biolistic transformation, and introduction of PPO and SPS gene constructs on performance of sugarcane clones in the field

Abstract

Stably transformed sugarcane plants were produced by the biolistic introduction of DNA into tissue-cultured cells. Constructs containing genes in sense and antisense orientation of polyphenol oxidase and sense orientation of sucrose phosphate synthase were used in the transformations. Regenerated plants were grown in a series of field experiments that incorporated commercial varieties, including Q117, from which the transgenic clones were derived and plants regenerated from tissue culture but not subjected to biolistic bombardment. In all experiments, the mean yield of transgenic sugarcane was lower than commercial varieties and the transgenic clones often exhibited lower sugar content, although individual transgenic clones in some experiments were not significantly different from Q117. Those plants regenerated from tissue culture but not bombarded were intermediate in their yield, and more clones were equivalent to Q117 in agronomic performance. Transformed plants produced by the bombardment of callus performed poorly but the results from the tissue-cultured controls indicated that not all of this could be due to somaclonal variation. Some aspect(s) of the process of transformation itself was deleterious and in most cases more significant than the effects due to tissue culture. Of the transgenic clones grown at Ayr, Queensland, 1.6% were equivalent to Q117 in sugar content and yield, suggesting that large numbers of transgenic clones would have to be generated using the current method in order to allow for selection of clones with acceptable agronomic performance.

Genetic Engineering of Saccharum

“…Improvement in transformation ef fi ciency was attributed to a higher embryogenic competence of in fl orescence tissue (Snyman et al 2006 ) . Besides improving transformation ef fi ciency, direct embryogenesis from leaf roll disks reduces the duration of exposure of explants to high levels of 2,4-D and minimizes the incidence of somaclonal variation (Snyman et al 2006 ) observed in transgenic plants generated from embryogenic callus (Gilbert et al 2005(Gilbert et al , 2009Vickers et al 2005b ) . However, one of the pitfalls of direct embryogenesis from leaf roll disks is the high incidence of escapes (Snyman et al 2000(Snyman et al , 2006 , possibly due to low surface area exposure of the embryogenic leaf roll disks to the selective agent during selection and regeneration.…”

“…Several studies in sugarcane have revealed the existence of cell and tissue cultureinduced somaclonal variation (Rajeswari et al 2009 ) as well as transformationassociated variation during biolistic (Gilbert et al 2005(Gilbert et al , 2009Vickers et al 2005b ) and Agrobacterium -mediated transformations (Carmona et al 2005 ) . Signi fi cant differences in agronomic performance of the transformed plants compared to the nontransformed parental variety were reported (Vickers et al 2005b ) and different genotyping techniques that included ampli fi ed fragment length polymorphism (Carmona et al 2005 ) , simple sequence repeat (Gilbert et al 2009 ) and random ampli fi cation of polymorphic DNA (Hussain 2005 ) were used to detect changes at the genomic level.…”

“…Signi fi cant differences in agronomic performance of the transformed plants compared to the nontransformed parental variety were reported (Vickers et al 2005b ) and different genotyping techniques that included ampli fi ed fragment length polymorphism (Carmona et al 2005 ) , simple sequence repeat (Gilbert et al 2009 ) and random ampli fi cation of polymorphic DNA (Hussain 2005 ) were used to detect changes at the genomic level. This indicates the need for further development of less aggressive transformation techniques and shorter exposure of transformed explants to in vitro culture conditions.…”

Rapid production of transgenic sugarcane with the introduction of simple loci following biolistic transfer of a minimal expression cassette and direct embryogenesis

“…An extended tissue culture period is required for the standard sugarcane transformation protocol in which embryogenic callus is the target tissue for gene transfer. Consequently, these protocols may promote the development of somaclonal variation, and less than optimal performance of many of the regenerated transgenic plants has been reported (Arencibia et al 1999;Gilbert et al 2005Gilbert et al , 2009Vickers et al 2005). Direct somatic embryogenesis (DSE; Desai et al 2004;Snyman et al 2006; Van Der Vyver 2010) from explants such as immature inflorescence, immature leaf whorl cross-sections containing immature inflorescence, and immature leaf whorl cross-sections have also been reported following biolistic gene transfer, but convincing molecular evidence for the transgenic nature of the events was not provided, and whole plasmids were used for gene transfer.…”

Cyanosiphovirus S-ESS1 Infecting Marine <i>Synechococcus</i> (<i>Chroococcales</i>) Almost Shows No Genetic Relationship to Known Cyanosiphoviruses

“…Sugarcane has been also genetically modified for sugar yield and quality traits (Botha and Groenewald 2001;Vickers et al, 2005), pharmaceuticals (Wang et al, 2005), novel sugars with potential benefits to consumer (OGTR, 2004). Besides, many biotic and abiotic stresses related to physiological characters have been studied in transgenic sugarcane.…”