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

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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.
Funding Information
  • Key Technologies Research and Development Program (2024YFD1200501)
  • National Natural Science Foundation of China (32530092)
  • China Agricultural Research System (CARS-26)
  • Citrus Research Board (5200-180)
  • National Institute of Food and Agriculture (2023-70029-41277)

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