Droplet-vitrification cryotherapy and thermotherapy as efficient tools for the eradication of apple chlorotic leaf spot virus and apple stem grooving virus from virus-infected quince in vitro cultures

Abstract

Viral diseases affect quince plant productivity and fruit quality. This study evaluated the effectiveness of droplet-vitrification cryotherapy and thermotherapy methods in the eradication of apple chlorotic leaf spot virus (ACLSV) and apple stem grooving virus (ASGV) from virus-infected in vitro cultures of quince rootstock ‘QA’ and cultivar ‘Neyshabour’. In vitro cultures infected with ACLSV and ASGV were thermo-treated at 38 °C for 0, 7, 10, and 15 days followed by apical shoot tip isolation. In the cryotherapy procedure, shoot tips were incubated on preculture medium and then exposed to plant vitrification solution 2 for 30 min at 0 °C prior to liquid nitrogen (LN) exposure. Shoot tips were warmed in unloading solution and placed on recovered medium. The frequency of virus eradication was determined using RT-PCR in plantlets recovered from thermo-and cryo-treatments and controls that were grown under in vitro conditions for 4 months. Droplet-vitrification cryotherapy and thermotherapy resulted in high frequencies of ACLSV and ASGV eradication from in vitro cultures of quince. The results showed that increasing the duration of thermotherapy significantly increased virus eradication. In vitro shoots of quince rootstock ‘QA’ and cultivar ‘Neyshabour’ heat-treated for 10 days were 64% and 67% free of ACLSV and 55% and 33% free of ASGV, respectively. All quince rootstock ‘QA’ plantlets regenerated from droplet-vitrification cryotherapy and RT-PCR assessed were free of ACLSV and 67% were free of ASGV. Cryotherapy and thermotherapy may be considered as promising methods for virus eradication programs in quince genotypes facilitating the production of healthy stock plants