No abstract

Five populations of lemon plants [Citrus limon (L.) Burm] obtained from undeveloped ovules through diVerent tissue culture procedures were examined for the presence of somaclonal and irradiationinduced genetic variation. Tested groups were: (1) nucellar seedlings; (2) organogenic, regenerated via adventitious buds from nucellar seedling internodes;(3) embryogenic population, regenerated from nonirradiated nucellar callus via somatic embryogenesis; (4) embryogenic population, regenerated from irradiated nucellar callus via somatic embryogenesis; and (5) protoplast-derived, regenerated via somatic embryogenesis. Genomic DNA samples from 360 plants (72 from each group) were screened for polymorphism among RAPD Wngerprints ampliWed by 10 decamer primers. Among all tested plants, genetic variation was detected only within the group of plants recovered from irradiated embryogenic calli. Out of 72 plants from that group, three had RAPD Wngerprints diVerent from the rest of the population, and fourth plant was found to be cytochimeric, consisting of diploid and tetraploid cells as revealed by Xow cytometry. In all other populations of regenerated plants, we did not come across any plants with changed ploidy level.

Citrus canker caused by Xanthomonas citri subsp. citri (Xcc) is a destructive citrus disease worldwide. Generating disease-resistant cultivars is the most effective, environmentally friendly and economic approach for disease control. However, citrus traditional breeding is lengthy and laborious. Here, we develop transgene-free canker-resistant Citrus sinensis lines in the T0 generation within 10 months through transformation of embryogenic protoplasts with Cas12a/crRNA ribonucleoprotein to edit the canker susceptibility gene CsLOB1. Among the 39 regenerated lines, 38 are biallelic/homozygous mutants, demonstrating a 97.4% biallelic/homozygous mutation rate. No off-target mutations are detected in the edited lines. Canker resistance of the cslob1-edited lines results from both abolishing canker symptoms and inhibiting Xcc growth. The transgene-free canker-resistant C. sinensis lines have received regulatory approval by USDA APHIS and are exempted from EPA regulation. This study provides a sustainable and efficient citrus canker control solution and presents an efficient transgene-free genome-editing strategy for citrus and other crops.

Transgenic plants of grapefruit (Citrus paradisiMacf.) cvs. 'Duncan', 'Flame', 'Marsh', and 'Ruby Red' were obtained using Agrobacterium tumefaciens-mediated transformation of seedling epicotyl tissue. Two citrus tristeza virus (CTV)-derived candidate resistance genes: '392' (3′ region of the p23 ORF plus 3′ untranslated region-UTR) and 'p23 hairpin' (sense-p23 ORF plus UTR plus antisensep23 ORF) were introduced into grapefruit using Agrobacterium strains EHA105 and EHA101, respectively. Epicotyl explants from 1-mo.-old in vitro etiolated seedlings were incubated in bacterial suspension. Green shoots that formed on explants after 4-5 wk after bacterial incubation were tested for the presence of the GUS gene by histochemical analysis. The percentage of GUS-positive shoots and transformation efficiency was 30.3±3.3% and 3.5% for treatment with EHA101 and 15.3±1.7% and 1.3% for treatment with EHA105. GUS-positive shoots were micrografted onto Carrizo citrange (Citrus sinensis L. Osbeck×Poncirus trifoliata L. Raf.) seedling rootstocks, and the presence of transgene sequences in these plants was confirmed by polymerase chain reaction (PCR), Southern blot, and reverse transcription PCR analyses. Resulting transgenic grapefruit plants were challenged with CTV and tobacco mosaic virus using a protoplast challenge assay as an initial screen to determine the effects of the transgenes on virus replication. Although complete RNA-mediated resistance was not achieved, preliminary results showed that 5.2% of the recovered transgenic plants containing the '392' CTV-derived sequence repeatedly exhibited reduced CTV replication in protoplasts. These plants are being further evaluated using the traditional method of virus inoculation followed by enzyme-linked immunosorbent assay.