Establishment of an <i>In Vitro</i> Propagation, Transformation, and Gene Editing System in Tomato (<i>Solanum lycopersicum</i>)

Abstract

Tomato (<em>Solanum lycopersicum</em>), a family of Solanaceae, is an annual crop grown in fields and greenhouses. Tomato is the fourth-largest agricultural crop in the world, with a market value of more than $50 billion. Numerous attempts were conducted on the tomato for further improvement via <em>in vitro</em> regeneration and transformation. This study aims to establish <em>in vitro</em> multiplication, transformation, and gene editing systems in tomatoes by using cotyledon explants. Cotyledon explants were placed on the MS medium supported with 6-benzyl adenine (BA) and kinetin (Kin) at four different concentrations for shoot induction. The shoot induction from cotyledon explants significantly increased, and the regeneration rate was 45% after using BA at 8.8 µM compared to Kin. For root induction, indole-3-butyric acid (IBA) at 4.8 µM concentration revealed the highest number of explants producing root (55%). Three <em>Agrobacterium tumefaciens</em> strains (EHA105, GV3101, and LBA4404) were used to establish and develop a tomato transformation method. Each <em>Agrobacterium</em> strain harbors the plasmid pCAMBIA2301, including the <em>nptII </em>marker. <em>Agrobacterium</em> strain LBA4404 showed the lowest number of dead explants, besides increasing the number of explant-produced shoots (30%) and the number of shoots per transformed explants compared to GV3101 and EHA105 strains. Our results revealed that the success of tomato transformation depends on selecting a suitable <em>A.</em> <em>tumefaciens</em> strain. Finally, the tomato gene editing system was successfully established by transferring the pCAMBIA1300-pCas9-sgRNA-U3 binary vector into the cotyledon using the selective <em>Agrobacterium</em> strain LBA4404, and the transformation effectivity was confirmed by PCR analysis.