Grapevine hairy root culture establishment, confirmation and maintenance protocol. v1

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A detailed protocol for establishing grapevine hairy root cultures via Rhizobium rhizogenes-mediated transformation of in vitro plantlets, adaptable to other species with minor adjustments to explant type or age.The protocol covers bacterial suspension preparation, infection procedure, hairy root confirmation, and subculturing. The protocol successfully established hairy roots from three Vitis vinifera cultivars (Chardonnay, Redglobe and Sultana), four Vitis rootstock hybrids (Gravesac, Ramsey, SO4 and 143B), and two other important South African plant species (Sutherlandia frutescens and Aspalathus linearis). In grapevine, hairy roots emerge from explants ~14 days post-infection, exhibit the characteristic non-geotropic, highly branched phenotype on hormone-free media, and continue robust development after excision. Genotypic confirmation employs PCR with newly developed universal multiplex primers compatible acrossR. rhizogenesstrains. Hairy roots can be subcultured indefinitely for biomass accumulation in bioreactors, and the protocol supports generation of CRISPR-edited roots.

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