Agrobacterium-mediated Genetic Transformation and Plant Regeneration from Cotyledons in Philippine Eggplant (Solanum melongena L.) Acc. ‘PH 11424’

Abstract

Eggplant (Solanum melongena L.) is one of the most important vegetables grown and consumed in the Philippines hence, continuous breeding programs are vital to maintain the supply of this economically important crop. This study demonstrates the first successful Agrobacterium-mediated genetic transformation and plant regeneration of the Philippine eggplant cultivar ‘PH 11424’, also known as ‘Mistisa’. Cotyledons from 2-wk-old seedlings were used as explants, which were transformed with disarmed Agrobacterium tumefaciens strain LBA4404 harboring a binary vector for CRISPR/Cas9 expression and hygromycin phosphotransferase (HPT), an antibiotic selection marker. Growth of shoot primordia from the agro-infected explants was observed during selective culture with 7.5 ppm hygromycin, which indicated an initial success in transformation. The putatively transformed shoot primordia were then transferred to an elongation medium. The elongated and hygromycin-resistant shoots were allowed to develop roots in hormone-free medium supplemented with hygromycin, and subsequently acclimatized under greenhouse conditions. The entire process took at least 5 – 6 mo. Of the total 585 agro-infected explants, the regeneration efficiency of rooted shoots was 4.6%. Successful transformation was confirmed by polymerase chain reaction (PCR) amplification of Cas9. Acclimatized plants tested positive for both transgenes. The surviving transgenic eggplants successfully reached maturity, flowered and set seed. These results demonstrate a working Agrobacterium-mediated transformation and plant regeneration protocols using cotyledons as explants in a Philippine eggplant genotype. These biotechnology tools are critical for the successful application of genetic engineering (GE) and new breeding techniques (NBTs) in eggplant crop improvement.