Elimination of the Crown Gall Pathogen, Agrobacterium vitis, from Systemically Infected Grapevines by Tissue Culture

Procedure for elimination of Agrobacterium vitis from systemically infected grapes using tissue culture therapeutics. (A) Shoot tips collected from actively growing mother plants that were established in the greenhouse from cuttings of crown gall-diseased Vitis vinifera cv. Riesling. (B) Shoot tip explants (2 to 3 cm in length) prepared for excision of buds and meristems by first removing large leaves near the apex (left) and then all leaflets (right). (C) Shoot apex including apical and adjacent axillary buds. (D) Apical bud (0.5 cm in length) from which leaves were excised, ready for establishment on solid tissue culture medium. (E) Close-up of an apical meristem (0.3 to 0.5 mm diam) dissected for establishment in tissue culture medium. (F) Axillary bud (0.5 cm in length) ready for establishment on solid tissue culture medium. (G) Different growth stages of apical and axillary buds after zero (left) to six (right) weeks of culture. (H) Meristem growing in liquid tissue culture medium on a filter paper bridge after 16 weeks in culture. (I) Plantlets were tested for A. vitis by magnetic capture hybridization in conjunction with real-time PCR (MCH-qPCR) after at least three subcultures onto fresh tissue culture medium. (J) Plantlets that tested negative for A. vitis were established in soil and grown in the greenhouse for further testing. (K) Actively growing plants in the greenhouse were tested for A. vitis using MCH-qPCR.