Vitrification–cryopreservation, an efficient method for eliminating Candidatus Liberobacter asiaticus, the citrus Huanglongbing pathogen, from in vitro adult shoot tips

Abstract

Huanglongbing disease (HLB), caused by Candidatus Liberobacter asiaticus, constitutes a most serious problem for the Chinese citrus industry. In this work, the use of vitrification-cryopreservation for eliminating Ca. L. asiaticus from naturally infected plants of several citrus species was investigated. Proliferating meristems were produced in vitro and excised tissue clumps were cryopreserved through vitrification using a plant vitrification solution 2. The health status of regenerated in vitro plants was checked by nested PCR. The putative HLB bacterial-free materials were subsequently re-tested after greenhouse acclimatization. Up to 98.1% of the plants obtained by cryopreservation were free from HLB bacterium, as compared with a sanitation rate of 25.3% yielded by conventional meristem tip culture. Light and electron microscopy observations of the meristem tips showed that the majority of the meristematic cells were injured either during the freezing/thawing step or during the osmotic dehydration step with plant vitrification solution 2. Only small areas of the meristematic dome survived the cryopreservation process, thereby increasing the probability of regenerating cells free of Ca. L. asiaticus. Large cells with big vacuoles and high water content, which are more likely to be infected by Ca. L. asiaticus, apparently cannot survive freezing in liquid nitrogen (LN). By contrast, small cells with dense cytoplasm located in the top layers of the meristem are more likely to escape invasion by Ca. L. asiaticus and can survive freezing in LN.

Eliminating Potato Virus Y (PVY) and Potato Leaf Roll Virus (PLRV) Using Cryotherapy of in vitro-grown Potato Shoot Tips

“…The best results for PVY (20.3%) and PLRV (23.2%) eradication and a 20.3% regeneration rate were found with the 0.3-mm meristem tips. This result is very similar to that reported by Ding et al (2008). Meristem size is a very critical factor for determining virus-free frequency in meristem culture.…”

“…As shown in Table 3, the highest eradication rate (98.3~99.1%) was found when the cryopreserved shoot tips were 1.0-1.5 mm in length and the frequency of PVY and PLRV eradication from regenerated plantlets was similar (96.4~99.3% and 97.3~ 99.7%, respectively), regardless of the shoot tip size. This result is very similar to that reported by Ding et al (2008). Although shoot tip size can be a critical factor in conventional virus elimination, this may not be the case with cryotherapy.…”

Potato Shoot Tip Cryopreservation. A Review

“…Cryotherapy works as a micro-scalpel, where larger, more hydrated cells outside the meristem die because of freezing, and only small cells within and close to the meristem survive cryopreservation (Benson 2008b;Helliot et al 2002). Cryotherapy has been successfully applied to eliminate or reduce viruses in banana (Helliot et al 2002), grapevine (Wang et al 2003), Prunus (Brison et al 1997), raspberry (Wang and Valkonen 2009b;Wang et al 2009), citrus (Ding et al 2008) and sweet potato (Wang and Valkonen 2008). Also potato shoot tips were freed from viruses by cryotherapy.…”

Biotechnology a Modern Tool for Fruits Production - A Review

“…Complete plants have been successfully regenerated from tissues cryopreserved at −196•C in liquid N 2 in several crops for several months to years (Ford et al, 2000;Panis et al, 2001;Halmagyi et al, 2004;Gupta and Reed, 2006;Ding et al, 2008). This method is now being practically used at several national and international germplasm banks.…”