High efficiency in planta transformation of Indian mustard (Brassica juncea) based on spraying of floral buds

Abstract

Brassica juncea (Indian mustard) and its sub-varieties include whole range of oilseed and vegetable mustard in China, Canada, Australia, Europe and other regions of the world. Application of genomics in varietal improvement of this crop has been limited in the past. Recently, published genome sequence has generated enormous amount of sequence information and set the stage for high-throughput functional genomics in this crop. However, high efficiency transformation has always been elusive and a major constraint in genetic improvement of this crop. Here, we report a method for high frequency in planta transformation of Indian mustard based on floral spray of Agrobacterium suspension. Successful genetic transformation was confirmed by polymerase chain reaction, reverse transcription quantitative PCR and assay for the reporter gene expression. The transformation efficiency was scored by PCR based analysis of transgene integration and expression in T1 plants. Transformation efficiency varied depending on the genotype, light conditions during co-cultivation and age of the experimental plant material. This method being simple and highly reproducible will be immensely beneficial for functional genomics and transgenic development programme in B. juncea. Efficiency of genetic transformation is elusive and a major constraint in Brassica juncea in spite of it being known as amenable species. It has been a major constraint for rapid functional genomics of genome sequence that has been recently published. An in-planta method has been developed which led to 30% transformation efficiency. It involved a typical mechanical maneuvering on the unopened buds before spraying of Agrobacterium suspensions.