Genetic engineering based on Agrobacterium-mediated transformation has been a desirable tool to manipulate single or multiple genes of existing genotypes of woody fruit crops, for which conventional breeding is a difficult and lengthy process due to heterozygosity, sexual incompatibility, juvenility, or a lack of natural sources. To date, successful transformation has been reported for many fruit crops. We review the major progress in genetic transformation of these fruit crops made in the past 5 years, emphasizing reproducible transformation protocols as well as the strategies that have been tested in fruit crops. While direct transformation of scion cultivars was mostly used for fruit quality improvement, biotic and abiotic tolerance, and functional gene analysis, transgrafting on genetically modified (GM) rootstocks showed a potential to produce non-GM fruit products. More recently, genome editing technology has demonstrated a potential for gene(s) manipulation of several fruit crops. However, substantial efforts are still needed to produce plants from gene-edited cells, for which tremendous challenge remains in the context of either cell’s recalcitrance to regeneration or inefficient gene-editing due to their polyploidy. We propose that effective transient transformation and efficient regeneration are the key for future utilization of genome editing technologies for improvement of fruit crops.

Citrus canker caused by Xanthomonas citri subsp. citri is one of the most devastating citrus diseases worldwide. Generating disease-resistant citrus varieties is considered one of the most efficient and environmentally friendly measures for controlling canker. X. citri subsp. citri causes canker symptoms by inducing the expression of canker susceptibility gene LOB1 via PthA4, a transcription activator-like (TAL) effector, by binding to the effector binding element (EBE) in the promoter region. In previous studies, canker-resistant plants were generated by mutating the coding region or the EBE of LOB1. However, homozygous or biallelic canker-resistant plants have not been generated for commercial citrus varieties, such as grapefruit (Citrus paradisi), which usually contain two alleles of LOB1 and thus, have two types of LOB1 promoter sequences: TI LOBP and TII LOBP. Two different sgRNAs were used to target both EBE types. Both 35S promoter and Yao promoter were used to drive the expression of SpCas9p to modify EBE_PthA4-LOBP in grapefruit. Using ‘Duncan’ grapefruit epicotyls as explants, 19 genome-edited grapefruit plants were generated with one biallelic mutant line (#Dun_Yao7). X. citri subsp. citri caused canker symptoms on wild-type and nonbiallelic mutant plants but not on #Dun_Yao7. XccPthA4 mutant containing the designer TAL effector dLOB1.5, which recognizes a conserved sequence in both wild-type and #Dun_Yao7, caused canker symptoms on both wild-type and #Dun_Yao7. No off-target mutations were detected in #Dun_Yao7. This study represents the first time that CRISPR-mediated genome editing has been successfully used to generate disease-resistant plants for ‘Duncan’ grapefruit, paving the way for using disease-resistant varieties to control canker.

Copper (Cu)-based fungicidal sprays are widely used on many crops although Cu sprays can be phytotoxic under some conditions. The mechanism of phytotoxicity is poorly understood but must involve toxic levels of Cu penetrating plant tissues. We studied the effect of different adjuvants on the deposition pattern of droplets and penetration of Cu (in Kocide fungicide) through isolated cuticles of ‘Marsh’ grapefruit leaves and ‘Valencia’ orange fruit. The addition of the silicone-based L-77 surfactant to the Kocide suspension markedly increased the spread of the droplets on cuticles and increased the penetration of Cu through fruit and abaxial leaf cuticles, both with stomatal pores, but not through astomatous adaxial leaf cuticles, which had much lower permeability. Urea and petroleum spray oil adjuvants had no effect on surface area of droplets or the penetration of Cu through leaf and fruit cuticles. Spray tank mixes of Cu fungicides with organosilicone surfactants should be avoided because these surfactants can enhance the penetration of Cu into citrus leaves and fruit thereby leading to phytotoxicity.