Using green fluorescent protein sheds light on Lupinus angustifolius L. transgenic shoot development

Abstract

Narrow-leaf lupin (NLL) is the main legume crop grown in rotation with wheat and other cereals in Western Australia. Efforts to improve NLL germplasm by use of genetic technologies have been hampered by the lack of an efficient genetic transformation method, an issue that is in common with dominant crop legumes globally. Prior research has primarily used the bar gene for phosphinothricin (PPT) resistance. The aim of recent research has been to investigate alternative selection methodologies, in order to determine whether the limitations of low frequency of transgenic shoots, combined with chimerism at T0 could be overcome. Investigation of hygromycin resistance as a selectable marker compared to PPT is reported here. The results suggested that hygromycin resistance was a more suitable selectable marker for NLL transformation than PPT. Surprisingly, from investigation of transformation using the GUS reporter gene, it was also observed that transformation frequency was greater when selection treatment was reduced or delayed, compared to the existing protocols. To further investigate this observation, an eGFP expressing construct was prepared. Observations within the first week after Agrobacterium exposure of lupin explants demonstrated that transformation of NLL explant cells was not a rate-limiting step. Instead, the results indicated that the current selection methodology was killing the cells that were competent to regenerate into transgenic shoots. It was concluded that further research on the development of the treated explants should focus on delayed selection and exposure to Agrobacterium of cells below the apical meristem.