Ron Gafny

Elimination of grapevine virus A (GVA) by cryopreservation of in vitro-grown shoot tips of Vitis vinifera L

Abstract Methods for the cryopreservation of in vitro-grown shoot tips of grapevine were recently developed [1] , [2] . The present study demonstrates that grapevine virus A (GVA) can be successfully eliminated from naturally infected grapevine by cryopreservation of in vitro-grown shoot tips. The various steps taken before freezing in liquid nitrogen did not, by themselves, eliminate GVA. However, the freezing step resulted in 97% GVA elimination. The size of the shoot tips used for cryopreservation influenced their survival rate, while viral eradication was independent of their size in the range of 0.5–2.0 mm. In comparison, plant regeneration from meristems increased with size, and meristems of 0.1 mm completely failed to regenerate. Regeneration from 0.4-mm meristems reached 100%, but none of the regenerated plantlets were GVA-free. Meristems of 0.2 mm resulted in only 12% GVA-free plants. Frequency of GVA elimination was not affected by the cryopreservation procedure, be it encapsulation–dehydration or vitrification. Leaf morphology of plants regenerated from cryopreserved shoot tips was similar to that from control shoot tips. Results from the present study suggest cryopreservation of shoot tips as a simple and efficient method for eliminating GVA from infected grapevine plants.

Cryopreservation of grapevine (Vitis vinifera L.) embryogenic cell suspensions by encapsulation-dehydration and subsequent plant regeneration

Embryogenic cell suspensions of grapevine (Vitis vinifera L.) were successfully cryopreserved by encapsulation-dehydration and subsequently regenerated into plants. Embryogenic cell suspensions were first-step precultured for 2 days in MGN liquid medium supplemented with increasing sucrose concentrations of 0.25, 0.5, 0.75 and 1 M. Precultured cells were then encapsulated and second-step precultured in MGN liquid medium containing 1 M sucrose for several days. Following preculture, encapsulated cells were dehydrated prior to direct immersion in liquid nitrogen for 1 h. After thawing, cryopreserved cells were post-cultured on MGN medium for survival. Surviving cells were then transferred to different media for regrowth and plant regeneration, respectively. An optimal viability of cryopreserved cells was achieved when encapsulated cells were dehydrated to 20.6% water content and subjected to 2–4 days of preculture on 1 M sucrose. MGN solid medium containing 2.5 g/l AC was found to promote viability of cryopreserved cells. Although cryopreserved cells showed a 5-day lag phase in regrowth, and their increase in fresh weight was only half that of control cells, their growth pattern was the same as that of control cells after two subcultures, following re-establishment in cell suspensions. Cryopreservation promoted embryogenesis and subsequent plant germination, compared with both the control and dehydration treatments. Leaf morphologies of plants regenerated from cryopreserved cells appeared to be similar to those of plants from control cells.

Cryopreservation of grapevine (Vitis spp.) embryogenic cell suspensions by encapsulation-vitrification

Embryogenic cell suspensions of two grapevine rootstocks: 110 Ritcher (V. berlandieri × V. rupestris), 41B (V. vinifera × V. berlandieri) and several table grape and wine cultivars (Vitis vinifera) were successfully cryopreserved by the encapsulation–vitrification method. Embryogenic cell suspensions were precultured for 3 days in liquid MGN medium supplemented with daily increasing sucrose concentrations of 0.25, 0.5, 0.75 M. Precultured cells were encapsulated and directly dehydrated with a highly concentrated vitrification solution prior to immersion in liquid nitrogen for 1 h. After rewarming at 40 °C for 3 min, cryopreserved cells were post-cultured on solid MGN medium supplemented with 2.5 g l−1 activated charcoal. Surviving cells were transferred to solid MGN medium for regrowth or solid MG medium for embryo development and then to solid WPM for plant regeneration. Optimal viability was 42–76% of cryopreserved cells when cell suspensions were precultured with a final sucrose concentration of 0.75 M and dehydrated with PVS2 at 0 °C for 270 min. Biochemical analysis showed that sucrose preculture caused changes in levels of total soluble protein and sugars in cell suspensions. Although the increase in fresh weight was significantly lower in cryopreserved cells than in control cells, the growth pattern of the cryopreserved cells and control cells was the same after two subcultures, following re-establishment in cell suspensions. Protocol developed in this study suggests a universal and highly efficient cryopreservation system suitable for several genetically diversed Vitis species.

Cryopreservation of in vitro-grown shoot tips of grapevine by encapsulation-dehydration.

In vitro-grown shoot tips of the LN33 hybrid (Vitis L.) and cv. Superior (Vitis vinifera L.) were successfully cryopreserved by encapsulation-dehydration. Encapsulated shoot tips were precultured stepwise on half-strength MS medium supplemented with increasing sucrose concentrations of 0.25, 0.5, 0.75 and 1.0 M for 4 days, with one day for each step. Following preculture, encapsulated shoot tips were dehydrated prior to direct immersion in liquid nitrogen for 1 h. After thawing, cryopreserved shoot tips were post-cultured on a post-culture medium for survival. An optimal survival of cryopreserved shoot tips was achieved when encapsulated shoot tips were dehydrated to 15.6 and 17.6% water content for the LN33 hybrid and cv. Superior, respectively. Comparison between the effects of dehydration with silica gel and by air drying on cryopreserved shoot tips, showed that survival was dependent on water content, not on dehydration method. The thawing method markedly affected survival of cryopreserved shoot tips, and thawing at 40 °C for 3 min was found best. No callus formation and fastest shoot elongation were obtained when cryopreserved shoot tips were post-cultured on the post-culture medium composed of half-strength MS supplemented with 1 mg l−1 BA and 0.1 mg l−1 NAA. With these optimized parameters, 60 and 40% survival of cryopreserved shoot tips were obtained for the LN33 hybrid and cv. Superior, respectively.

Nucleotide sequence of the coat protein gene of citrus tristeza virus: comparison of biologically diverse isolates collected in Israel

The sequences of the coat protein genes of four seedling yellows (SY) and four non-SY (NSY) of citrus tristeza virus (CTV) isolates, which were collected in Israel over a period of 30 years, were analyzed. Pairwise comparisons showed extensive similarities in the nucleotide and amino acid sequences of six isolates designated the VT group. This group consists of three NSY isolates that cause a very mild CTV reaction on the sensitive combination of sweet orange (SwO) grafted on sour orange (SO), and three SY isolates that cause severe SY and SwO/SO reactions. MT, a CTV isolate that is consistently nontransmitted byAphis gossypii, was found to be different in two amino acids (Val 103 and Glu 113) from each of theA. gossypii transmissible CTV isolates. Sequencing of the cDNA clones obtained from ST, a variably transmitted CTV isolate, showed extensive sequence variation among the tested clones. The sequence information indicates that the current CTV epidemics in Israel are caused by at least two CTV subspecies (VT and HT) displaying extensive differences in their coat protein genes.

Serological detection of grapevine virus A using antiserum to a nonstructural protein, the putative movement protein.

ABSTRACT Grapevine virus A (GVA) is implicated in the etiology of the rugose wood disease. The coat protein (CP) and the putative movement protein (MP) genes of GVA were cloned and expressed in Escherichia coli and used to produce antisera. Both the CP and the MP were detected with their corresponding antisera in GVA-infected Nicotiana benthamiana. The MP was first detected at an early stage of the infection, 6 to 12 h after inoculation, and the CP was detected 2 to 3 days after inoculation. The CP and MP were detected by immunoblot analysis in rugose wood-affected grapevines. The MP could be detected in GVA-infected grapevines that tested negative for CP, both with CP antiserum and with a commercially available enzyme-linked immunosorbent assay kit. The study shows that detection of the nonstructural MP may be an effective means for serological detection of GVA infection in grapevines.

Functional analysis of the grapevine virus A genome.

Grapevine virus A (GVA) carries five open reading frames (ORFs). Only the coat protein ORF has been experimentally identified as such; the roles of some of the other ORFs have been deduced by sequence homology to known genes (Minafra et al., 1997). The construction of a full-length, infectious clone of GVA has been previously reported. In an attempt to experimentally define the role of the various genes of GVA, we utilized the infectious clone, inserted mutations in every ORF, and studied the effect on viral replication, gene expression, symptoms and viral movement. Mutations in ORF 1 abolished RNA replication. Mutations in ORF 2 did not affect any of the aforementioned parameters. Mutations in ORFs 3 and 4 restricted viral movement. Mutations in ORF 5 rendered the virus asymptomatic, and partially restricted its movement.

D-RNA Molecules Associated with Subisolates of the VT Strain of Citrus Tristeza Virus which Induce Different Seedling-Yellows Reactions

Citrus tristeza virus (CTV) strains were previously catalogued as seedling-yellows (SY) and non-SY (nSY) types, according to their yellowing and stunting effects on indicator seedlings. Among subisolates of the VT strain, which were selected from chronically infected Alemow plants, there was a correlation between the presence of 2.4-, 2.7- and 4.5-kb D-RNAs, and SY and nSY reactions, respectively. Similarly, plants infected with Mor-T subisolates, which cause SY, contained D-RNAs of 2.6 to 2.8 kb, while nSY subisolates from recovered sour orange tissue contained a major D-RNA of 5.1 kb. Plants harboring the 2.7-kb D-RNA were protected against challenge inoculation with a subisolate harboring the 4.5-kb D-RNA. This study suggests that the nSY reaction results either from the absence of SY gene(s) in the genomes of certain CTV strains or through the suppression of the effects of SY gene(s) by D-RNAs with 5′ parts larger than 4000 nt.

Infectious RNA Transcripts from Grapevine Virus A cDNA Clone

A full length cDNA clone of grapevine virus A (GVA) was constructed downstream from the bacteriophage T7 RNA polymerase promoter. Capped in vitro-transcribed RNA was infectious in Nicotiana benthamiana and N. clevelandii plants. Symptoms induced by the RNA transcripts or by the parental virus were indistinguishable. The infectivity of the in vitro-transcribed RNA was confirmed by serological detection of the virus coat and movement proteins and by observation of virions by electron microscopy. This is the first report of infectious RNA transcripts derived from a full-length cDNA clone of a member of the Vitivirus genus.

Transgenic Nicotiana benthamiana and grapevine plants transformed with grapevine virus A (GVA) sequences

As part of a project to construct virus-resistant transgenic grapevines, grapevine virus A (GVA) coat protein (CP) sequences were introduced into grapevine and the model plantNicotiana benthamiana. The engineered constructs were tested for their ability to engender resistance inN. benthamiana. A number of GVA-CP-transformedN. benthamiana lines have become resistant to GVA. Preliminary biological and molecular analyses strongly suggest that in most cases the obtained resistance is RNA-mediated. Several lines of transgenic grapevine were obtained.