A rapid and broadly applicable in planta transformation and genome editing system involving wounded epicotyls in citrus

Abstract

Genetic transformation in citrus has largely relied on in vitro tissue culture-based approaches, which are labor intensive, time consuming, and strongly affected by genotype-dependent regeneration capabilities. In this study, we developed a rapid and broadly applicable in planta transformation system using wounded citrus epicotyls. Agrobacterium, plant growth regulators, and antibiotics were sequentially delivered to wounded epicotyls through localized agarose-based chambers to facilitate transformation, regeneration, and selection. The optimal combination of 3.0 mg/L 6-benzylaminopurine and 0.05 mg/L 1-naphthaleneacetic acid resulted in a nonchimeric transformation efficiency of 13.92% in Carrizo citrange. The system was further validated in 5 additional citrus genotypes, including sweet orange, pummelo, lemon, Citrus junos, and kumquat, with nonchimeric transformation efficiencies ranging from 2.58% to 18.68%. Transgenic plants were obtained within approximately 40 d after seed sowing. Furthermore, the system supported efficient CRISPR/Cas9-mediated genome editing and enabled the recovery of a putative transgene-free LATE MERISTEM IDENTITY1-edited plant exhibiting an oil glandless phenotype. This rapid and efficient platform provides a valuable tool for functional genomics and genome editing in citrus and may facilitate genetic improvement in other perennial woody species.