CRISPR/Cas12a-Mediated Knockout of INNER NO OUTER (INO) Gene in Musa balbisiana cv. Bhimkol

Abstract Banana ( Musa spp.) is a vital staple food and cash crop cultivated in over 140 countries, providing nourishment and livelihoods to more than 400 million people worldwide. In this context, Bhimkol ( Musa balbisiana , BB genome), a diploid banana variety native to Northeast India holds significant nutritional and commercial value. Its high iron and nutrient content have already been commercially validated through products like Bhimvita and Bhimshakti, which utilize fresh fruit pulp as nutrient-rich food for infants. However, Bhimkol fruits typically contain 100–150 seeds, an undesirable trait for product development. The manual removal of these seeds significantly increases production time and labour costs. Furthermore, because bananas are recalcitrant to traditional breeding, there is a constant need for rapid in vitro transformation protocols. To address these challenges, as a proof of concept, our research aims to knockout the INNER NO OUTER ( INO ) gene, which is responsible for ovule development. Using CRISPR/Cas12a technology, we established an efficient and reproducible in vitro regeneration and transformation system using Embryogenic Cell Suspensions (ECS). The resulting CRISPR-edited plantlets exhibited various mutations, including insertions and deletions (INDELs) within the targeted INO gene. These INDELs resulted in frameshift mutations that triggered premature stop codons. While these genetic changes are expected to render the banana seedless, phenotypic verification is currently underway to confirm the absence of seeds in mature fruit. Significance Statement Despite its superior nutritional profile, the commercial viability of the Bhimkol banana ( Musa balbisiana ) is restricted due to abundance of seeds (100–150 per fruit). This study employs CRISPR/Cas12a-mediated knockout the INNER NO OUTER ( INO ) gene in Bhimkol and expected to develop seedless fruits. The resulting plantlets exhibit targeted indels that trigger frameshift mutations, effectively disrupting ovule developmental INO gene.