In planta transformation in wheat: an improved protocol to develop wheat transformants.

Background: Lack of efficient transformation protocol continues to be a major bottleneck for successful genome editing or transgenic development in wheat. An in planta transformation method was developed in Indian bread wheat in earlier study (Vasil et al. in Nat Biotechnol 10:667-674, 1992) which was labour-intensive and time-consuming. In the present study, in planta transformation method was improved to make it simple, efficient, less labour-intensive and time-saving.

Methods And Results: PCR-based screening for generated transformants at T stage was introduced in this method. Shoot apical meristem of two days old wheat seedling was inoculated with the routine active culture of Agrobacterium tumefaciens harboring plasmid pCAMBIA1300-Ubi-GFP having gene GFP under the control of Zea mays ubiquitin promoter. PCR analysis at T stage confirmed 27 plants to be transgene positive. These 27 plants were only taken to the next generation (T) and the rest were discarded. At T generation 6 plants were analyzed to be PCR positive. Out of them, 4 plants were confirmed to have stable integration of transgene (GFP). Fluorescent microscopy at T stage confirmed the 4 Southern hybridization positive plants to be expressing reporter gene GFP.

Conclusions: Screening at T stage, reduced the load of plants to be taken to T generation and their screening thereof at T with no overall loss in transformation efficiency. We successfully transformed wheat genotype HD2894 with 3.33% transformation efficiency using a simple, effective method which was less labour-intensive and less time-consuming. This method may be utilized to develop wheat transgenic as well as genome edited lines for desirable traits.