Development of efficient embryo-derived regeneration system and optimization of genetic transformation in cumin (Cuminum cyminum L.)

Cumin (Cuminum cyminum L.) is an important spice crop with high agronomic and economic importance. A direct regeneration system using embryogenic explants in cumin was established to develop a highly efficient transformation system. Cumin embryos were utilized as an explant which shows higher regeneration efficiency on Gamborg's B5 media supplemented with 2.0 mu M BA+0.5 mu M NAA. Transformation of pSIM24-eGFP plasmid in cumin was carried out through Agrobacterium tumefaciens EHA105 and biolistic gene gun method. The transgenic explants were confirmed for green fluorescent protein (GFP) gene integration through PCR analysis. The Agrobacterium-mediated transformed explants showed higher regeneration and transient transformation efficiency with 0.5 OD600 of cell density and 24 h of co-cultivation compared to 0.4 OD600 with 24 h, 48 h, and 72 h co-cultivation time and 0.5 OD600 with 48 and 72 h co-cultivation time. It was further confirmed by GFP expression analysis through real-time PCR. Gene gun-mediated transformed explants were cultured on different osmolytes (mannitol, sorbitol, and sucrose) containing media to reduce bombardment stress on explants. Compared to mannitol and sucrose-containing media, transformed explants cultured on sorbitol- containing media showed higher rates of regeneration and transformation. These results were further confirmed by real-time PCR analysis as prominent GFP expression was found in explants cultured on sorbitol-containing media compared to other osmolytes containing media. In the current study, we have developed an efficient transient transformation system with higher gene expression and regeneration efficiency.