Attempts to obtain Huanglongbing resistant or tolerant sweet orange by embryo rescue from healthy chimeras of diseased citrus fruit

Huanglongbing remains a threat in the cooler citrus production areas of South Africa despite restrictions on the movement of citrus material from infected areas, cultural control measures, planting of healthy certified trees and the eradication of infected material. The ultimate control strategy would be the use of resistant plant material. Attempts to obtain resistance with conventional breeding have been unsuccessful. A new approach is to utilize embryo rescue of seed from healthy chimera sections of diseased fruit. The embryos were obtained from wide, asymptomatic sections of symptomatic fruit and cultured on Murashige and Tucker medium. Once the embryos developed to approximately 1 cm, they were micro-grafted to virus-free rootstocks. After further development to 10 to 15 mature buds, they were multiplied on virus-free rootstocks by budding. Growing shoots were challenged with "Candidatus Liberibacter africanus" via the psylla insect vector, Trioza erytreae Del G. for seven days on a young shoot. Afterwards, the psylla were collected from the plants and tested by polymerase chain reaction (PCR) and dot blot hybridisation, using the primer pair A2/J5, or probes to the amplicon, to determine if the insects used were infectious. Three months after the Liberibacter challenge, the same primer pair was used to evaluate the plants by PCR for the presence of the bacterium. Two clones remained negative after challenge, but the ultimate evaluation will be in the field where they will be exposed to repeated natural infection by the insect vector.