Production of Lily Symptomless Virus-free Plants by Shoot Meristem Tip Culture and In Vitro Thermotherapy

Abstract

<jats:p>To produce virus-free plants, a simple and original protocol was established by combining several techniques: repeated shoot meristem excision before and during in vitro culture and thermotherapy applied to bulblets in vitro. Lily symptomless virus (LSV) is a major virus that decreases plant growth vigor and the quality of cut flowers, yet infected plants show no distinct symptoms. Stock bulbs of pollenless Asiatic hybrid lily (<jats:italic>L. × elegans</jats:italic> Thunb) lines (‘409’ and ‘599’) were used as explant. Shoot meristems were excised and micropropagated. Thermotherapy (42 days at 35 °C) was applied to in vitro growing bulblets and a second meristem cut was then made from heat-treated material. Leaf tissues from bulblets formed before or postheat treatments were analyzed either by enzyme-linked immunosorbent assay or by reverse transcriptase–polymerase chain reaction. Line ‘499’ produced LSV-free plants without heat treatment, but line ‘599’ produced LSV-free plants only after heat treatment. The virus-free lily bulblets grew vigorously in vitro and acclimatized promptly. It is suggested that thermotherapy given to in vitro growing bulblets effectively eliminated the virus and induced a fast and efficient micropropagation technique for virus-free mother plant stock.</jats:p>

Disinfection of explants for saffron (Crocus sativus) tissue culture

“…The disinfection of biological material, however, is much more difficult and requires greater consideration. In plants, in theory, only the shoot apical meristem is free from any endophytes and viruses (Nesi et al 2009). This is because apical meristems are an actively diving group of cells without a well-established vascular system but their plasmodesmata are too small to allow the cell-to-cell movement of endophytes; in addition, virus replication (i.e., the viral inactivating system) cannot cope with rapid meristem cell division, and a low water concentration and a high osmotic pressure, or high auxin levels might also limit endophytes (Elmi, West 1995;Everett 2006;Alam et al 2013).…”

“…Microorganisms are not the only problem while establishing a micropropagation protocol. Bacteria and fungi can be removed during the initiation of in vitro cultures from standard disinfection of organs, but viruses [which are a serious threat with vegetatively propagated geophytes (Nesi et al 2009), decreasing plant growth vigour] cannot. The detection of viral agents in micropropagated geophytes can be achieved by enzyme-linked immunosorbent assay (ELISA) or by more sensitive polymerase chain reaction (PCR) techniques (Dorrigiv et al 2013;Nesi et al 2013).…”

“…Viruses can be eliminated by heat treatments (thermotherapy; e.g. 30 to 40 days at 35 °C) of in vivo-or in vitro-cultured plants and bulbs/bulblets and/or meristem isolation (Nesi et al 2009).…”

RT‐PCR tests for sensitive detection of the major Ranunculus‐infecting viruses: field and in vitro applications

“…Shoot apical meristem culture is considered a successful method for virus‐free rescue of valuable plant clones, at least for several virus infections (Fraga et al ., ; Balukievicz & Kryczynski, ; Ram et al ., ; Nesi et al ., ; Milošević et al ., ). Here, a new protocol for in vitro culture associated to SAM was devised that was tailored for ranunculus species.…”

“…(Balukievicz & Kryczynski, ; Ram et al ., ), Lilium spp. (Nesi et al ., ) and Impatiens spp. (Milošević et al ., ); however, this technique has still not been reported for ranunculus.…”