Biology subjects

Sagarbarria, M. G. S.

Publications and source records attributed to Sagarbarria, M. G. S..

2 recordsLinked to original sources

bioRxiv2023

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

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 a Philippine eggplant cultivar, PH 11424 also known as Mistisa. Cotyledons from two-week 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 transformation success. 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 months. Of the total 585 agro-infected explants, the regeneration efficiency of rooted shoots was 5.1%. Successful transformation was confirmed by polymerase chain reaction (PCR) amplification of Cas9. Acclimatized plants tested positive for the transgene. The 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.

plant biology↗

bioRxiv2023

Usefulness of Current sgRNA Design Guidelines and in vitro Cleavage Assays for Plant CRISPR/Cas Genome Editing: A Case in Eggplant (Solanum melongena L.)

The advent of genome editing platforms such as the CRISPR/Cas9 system ushers an unprecedented speed on the development of new crop varieties that can withstand agricultural challenges of the 21st century. The CRISPR/Cas9 system depends on the specificity of engineered single guide RNAs (sgRNAs). However, sgRNA design in plants can be challenging due to a multitude of design tools to choose from, many of which use guidelines that are based on animal experiments yet allow the use of plant genomes. Upon choosing sgRNAs, it is also unclear whether an in vitro assay is needed to validate the targeting efficiency of a particular sgRNA prior to in vivo delivery of the CRISPR/Cas9 system. Here, we demonstrate the in vitro and in vivo activity of four different sgRNAs that we selected based on their ability to target multiple members of the eggplant polyphenol oxidase gene family. Some sgRNAs that have high in vitro cleavage activity did not produce edits in vivo, suggesting that an in vitro assay may not be a reliable basis to predict sgRNAs with highly efficient in vivo cleavage activity. Further analysis of our sgRNAs using other design algorithms suggest that plant-validated criteria such as the presence of necessary secondary structures and appropriate base-pairing may be the reason for the discrepancy between our observed in vitro and in vivo cleavage efficiencies. However, recent reports and our data suggests that there is no guaranteed way to ensure in vivo cleavage of chosen sgRNAs. Key MessageO_LIin vitro cleavage assay of sgRNAs was able to identify low activity sgRNAs but did not 13 reliably predict in vivo mutagenesis. C_LIO_LIUsing multiple sgRNAs that meet the plant-validated parameters and have high activity in vitro in plant genome editing is critical to ensure success. C_LI

molecular biology↗