Abiotic stress improves in vitro biological indexing ofGrapevine leafroll-associated virus-3in red grapevine cultivars

Abstract

Background and Aims:Virus detection is necessary for in vitro production and propagation of virus-free plants, for in vitro conservation and when material is received from overseas in tissue culture. In vitro biological virus indexing is among the reliable techniques. The aim of the present study was to develop an in vitro graft-free protocol for indexing of Grapevine leafroll-associated virus-3 (GLRaV-3). Methods and Results: In vitro GLRaV-3-infected shoots were cultured on half-strength Murashige and Skoog medium supplemented with either 150 mmol NaCl or 4% polyethylene glycol (PEG) 8000 to induce development of the virus symptoms. After 4 weeks of culture, the proportion of the symptom expression was 86 and 81% for Cabernet Sauvignon, 100 for '6-12-2', 78 and 72% for Red Globe and 70 and 60% for Kyoho in the diseased shoots stressed by 150 mmol NaCl and 4% PEG, respectively. Expression of virus symptoms was more obvious and reliable when in vitro plantlets were stressed by NaCl than by PEG. Conclusions: Salt and drought stress induced by NaCl and PEG 8000 can improve in vitro biological indexing of GLRaV-3 in red grapevine cultivars. Significance of the Study: The techniques developed in the present study would have potential application to GLRaV-3 indexing of red grapevine cultivars.

Development, histological observations and Grapevine leafroll‐associated virus‐3 localisation in in vitro grapevine micrografts

“…The pH of the medium was adjusted to 5.8 prior to autoclaving at 121°C for 20 min, as described by Cui et al . (). The cultures were grown at a temperature of 24 ± 2°C under a 16‐h photoperiod with a light intensity of 50 µM m −2 s −1 provided by cool‐white fluorescent tubes.…”

“…Reverse transcription‐polymerase chain reaction analysis for GLRaV‐3 was conducted in in vitro stock shoots at the beginning of the experiments, and in upper or lower parts of micrografts of healthy scions or rootstocks at different points of time of micrografting, according to Cui et al . (). Total RNA was extracted from samples (0.5 g fresh weight) using the Trizol Reagent (Invitrogen, Carlsbad, CA, USA), according to the manufacturer's instructions; cDNA was synthesised on 30 µg of total RNA using recombinant Moloney murine leukaemia virus (MMLV) reverse transcriptase (Promega, Madison, WI, USA), according to the manufacturer's instructions.…”

“…Grapevine red wine cultivar ‘Cabernet Sauvignon’ ( Vitis vinifera ), which is susceptible to GLRaV‐3 and usually used as an indicator for this virus (Rowhani et al ., ; Cui et al ., ), was used in this study. Both healthy and GLRaV‐3 infected in vitro stock shoots were established according to Cui et al .…”

“…Both healthy and GLRaV‐3 infected in vitro stock shoots were established according to Cui et al . (, ) and maintained on a shoot maintaining medium (SMM) containing half‐strength Murashige & Skoog () medium (MS) supplemented with 30 g L −1 sucrose and 7 g L −1 agar. The pH of the medium was adjusted to 5.8 prior to autoclaving at 121°C for 20 min, as described by Cui et al .…”

“…In addition to the more traditional serological methods like enzyme‐linked immunosorbent assay (ELISA, Martelli & Walter, ; Martelli, ; Maree et al ., ), molecular analysis like reverse transcription‐polymerase chain reaction (RT‐PCR, Rowhani et al ., ; Maree et al ., ), and more advanced technique like next‐generation sequencing (NGS, Maree et al ., ; Barba et al ., ; Rwahnih et al ., ), biological indexing is among the reliable methods used for virus diagnosing (Tanne et al ., ; Martelli & Walter, , Valat et al ., ; Vidalakis et al ., ; Rowhani et al ., ; Pathirana & McKenzie, 2005; Kapari‐Isaia et al ., ; Wang & Valkonen, ; Maree et al ., ; Constable et al ., ; Cui et al ., ). In vitro virus indexing can avoid potential interferences such as environments, nutrients and other pathogens that are not studied in a given research, which exist in the field or greenhouse (Valat et al ., ; Constable et al ., ; Cui et al ., ). Compared with the traditional in vivo grafting, in vitro micrografting is time‐ and labour‐saving, and efficient (Tanne et al ., ; Valat et al ., ; Pathirana et al ., 2005; Kapari‐Isaia et al ., ; Wang & Valkonen, ).…”