Tissue-Culture-Independent Recovery of GFP-Positive and MYC2-Edited Calamansi (Citrus microcarpa) Shoots

Genotype-dependent regeneration limits genetic transformation in citrus. We adapted a localized Agrobacterium-mediated in planta transformation procedure to recover green fluorescent protein (GFP)-positive and MYC2-edited shoots from juvenile calamansi without conventional in vitro tissue culture. Seedlings were inoculated 18–20 d after germination, and GFP-positive shoots were identifiable approximately 30 d later. Four independent experiments using a GFP-marked empty editing vector yielded 49 GFP-positive seedlings among 384 inoculated seedlings. GFP-positive seedling frequencies ranged from 5.2% to 18.8% (mean ± standard deviation, 12.8 ± 5.7%); 33 seedlings produced uniformly fluorescent shoots. In a separate experiment using a clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) construct targeting MYC2, 13 GFP-positive shoots were recovered from 192 inoculated seedlings (6.8%). All 13 shoots yielded a GFP-specific polymerase chain reaction (PCR) product, and target-site amplicon sequencing detected mutations in every sample. Edited reads accounted for 82.8–99.5% of quality-filtered reads in individual samples. Under our production conditions, the year-round availability of polyembryonic calamansi seeds provided a regular supply of juvenile seedlings for inoculation. The procedure provides edited calamansi material for subsequent genetic and functional characterization. Our protocol was successfully applied to Shatian pummelo, as demonstrated by fluorescence screening and GFP-specific PCR results from a single preliminary trial.