Grapevine red blotch virus (GRBV) has a monopartite single-stranded DNA genome and is the type species of the genus Grablovirus in the family Geminiviridae. To address the etiological role of GRBV in the recently recognized red blotch disease of grapevine, infectious GRBV clones were engineered from the genome of each of the two previously identified phylogenetic clades for Agrobacterium tumefaciens-mediated inoculations of tissue culture-grown Vitis spp. plants. Following agroinoculation and one or two dormancy cycles, systemic GRBV infection was detected by multiplex polymerase chain reaction (PCR) in Vitis vinifera exhibiting foliar disease symptoms but not in asymptomatic vines. Infected rootstock genotype SO4 (V. berlandieri × V. riparia) exhibited leaf chlorosis and cupping, while infection was asymptomatic in agroinoculated 110R (V. berlandieri × V. rupestris), 3309C (V. riparia × V. rupestris), and V. rupestris. Spliced GRBV transcripts of the replicase-associated protein coding region accumulated in leaves of agroinfected vines, as shown by reverse-transcription PCR; this was consistent with systemic infection resulting from virus replication. Additionally, a virus progeny identical in nucleotide sequence to the infectious GRBV clones was recovered from agroinfected vines by rolling circle amplification, cloning, and sequencing. Concomitantly, subjecting naturally infected grapevines to microshoot tip culture resulted in an asymptomatic plant progeny that tested negative for GRBV in multiplex PCR. Altogether, our agroinoculation and therapeutic experiments fulfilled Koch's postulates and revealed the causative role of GRBV in red blotch disease.
Several factors that affect in vitro establishment, proliferation, and rooting of thirteen Malus cultivars and rootstocks were studied . Apple shoot tips (1 .5 ± 0.5 cm in length) were established using ascorbic and citric acids as antioxidants . Four proliferation media containing 1 .0 mg 1 -1 BA and different concentrations of IBA and GA3 were tested. Proliferation rates varied depending on the genotype and medium used . The highest proliferation rate was obtained for a rootstock that produced 11 .6 ± 2 .5 shoots (1 .5 ± 0 .8 cm in length) per tube per month . Rooting was induced with IBA for all the genotypes tested . The optimal IBA concentration was cultivar dependent (between 0 .1 and 1 .0 mg 1-1 IBA), and lower concentrations were necessary to induce rooting in liquid rather than in solid medium .The effects on shoot-tip proliferation of cefotaxime, carbenicillin and kanamycin, three antibiotics commonly used for transformation studies, were also evaluated . Cefotaxime at 200 mg 1 -1 stimulated shoot growth and development, but at 500 mg 1 -1 caused abnormal shoot morphology. Carbenicillin at 500 mg 1 -1 , alone or in combination with cefotaxime at 200 mg 1 -1 , inhibited proliferation and caused excessive enlargement of the basal leaves, inducing callus formation and release of phenolic compounds in the medium . Kanamycin at 50 mg 1 -1 was phytotoxic and caused shoot chlorosis and necrosis . Consideration of the toxicity of these antibiotics is critical when designing transformation schemes for selection and recovery of transgenic apple plants .Abbreviations: BA = benzyladenine ; cef = cefotaxime ; crb = carbenicillin ; GA3 = gibberellic acid ; IBA = indole-3-butyric acid ; Kan = kanamycin ; ms = Murashige and Skoog [19] macro-and micro-nutrients ; NAA = naphthalene-acetic acid . 55
In vitro regeneration and biolistic transformation procedures were developed for several commercial chrysanthemum Dendranthema grandiflora Tzvelev, syn. Chrysanthemum morifolium Ramat. cultivars using leaf and stem explants. Studies on the effect of several growth regulators and kanamycin on chrysanthemum regeneration were conducted, and a step-wise procedure to optimize kanamycin selection and recovery of transgenic plants was developed. A population of putative transformed chrysanthemum plants cvs. Blush, Dark Bronze Charm, Iridon, and Tara, was obtained after bombardment with tungsten microprojectiles coated with the binary plasmid pBIN19 containing the nucleocapsid (N) gene of tomato spotted wilt virus (TSWV) and the marker gene neomycin phosphotransferase (NPT II). PCR analysis of 82 putative transgenic plants selected on kanamycin indicated that the majority of the lines (89%) were transformed and contained both genes (71%). However, some transgenic lines contained only one of the genes: either the NPT II (15%) or the TSWV (N) gene (14%). Southern blot analysis on selected transgenic lines confirmed the integration of the TSWV (N) gene into the chrysanthemum genome. These results demonstrate the development of an efficient procedure to transfer genetic material into the chrysanthemum genome and selectively regenerate transgenic chrysanthemum plants at frequencies higher than previously reported.
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