Gene stacking in 1-year-cycling APETALA1 citrus plants for a rapid evaluation of transgenic traits in reproductive tissues

Rapid flowering is crucial to perform functional genomic studies to investigate reproductive biology characteristics and fruit quality-related traits in fruit trees. However, long generation cycles of woody plants considerably delay this evaluation. Through genetic transformation, juvenile periods can be significantly shortened by overexpression of flower meristem-identity genes. Transgenic APETALA1 (AP1) citrus plants behave as rapid-cycling trees, since 1-year-old seedlings promptly show precocious flowering and fruiting. By transgene stacking into these short-generation AP1 and nptII/GUS-positive plants, expression of novel transgenes could theoretically be examined as quickly as 1 year after retransformation. Establishment of the selection and regeneration conditions for the production of retransformed individuals with marker genes is detailed in this communication. Hpt and bar genes were used as the second selectable marker genes. PCR and Southern blot analyses confirmed the recovery of retransformed shoots. AP1 transcript accumulation and GUS and GFP expression were assessed in leaves, and flowers and fruit organs of rapid-cycling retransformed lines, respectively, as early as 1 year after plant generation and during three consecutive years, demonstrating that the principle of stable transgene stacking on early-fruiting transgenic trees is feasible.