Transient transformation of sweet cherry pollen with a hairpin gene construct targeting S haplotype-specific F-box, the pollen S determinant of Prunus self-incompatibility

Sweet cherry (Prunus avium) exhibits gametophytic self-incompatibility (GSI), which inhibits fertilization from self-pollen and pollen containing an S-allele that matches that in the maternal parent. Although several sweet cherry mutants with pollen-part self-compatibility (SC) exist, it is still difficult to break through self-incompatibility (SI), artificially. The pollen determinant gene, S haplotype-specific F-box (SFB), is suggested to be indispensable for self-pollen rejection in Prunus GSI. In the present study, we attempted to confer SC on sweet cherry pollen by silencing PavSFB. A hairpin gene sequence, hpPav6, was designed from a 320-bp fragment of PavSFB-S⁶ and Pdk intron. Ectopic expression of hpPav6 reduced the transcript level of PavSFB-S⁶ to half in the agroinfiltrated tobacco leaves, which indicated its effectiveness to silence PavSFB-S⁶. A plasmid containing LAT52 promoter-driven hpPav6 and beta-glucuronidase (GUS) was constructed to transform pollen using particle bombardment. Particle bombardment of the constructed plasmid induced GUS expression in ∼1% 'Satonishiki' (S³S⁶) pollen using an optimized parameter-set. A small particle size (0.6 μm) and high bombarding pressure (1550 psi) tended to improve the transformation efficiency. The bombarded pollen was used for self-pollination; however, clear evidence indicating transformation to SC was not obtained. The tubes of the bombarded pollen reached near the base of the self-style in two out of the 93 tested pistils; however, no fruit set was obtained in over 500 flowers pollinated on the plant.