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Abstract

Shallot (Allium cepa var. aggregatum) is an important vegetable crop belonging to the genus Allium. The present study attempted to develop an efficient droplet-vitrification cryopreservation method for shallot ‘10603’ shoot tips. In vitro stock shoots were maintained on Murashige and Skoog (1962) medium (MS) supplemented with 30 g L-1 sucrose, 0.5 mg L-1 BAP, 0.1 mg L-1 NAA and 8 g L-1 agar (pH=5.8). Shoot tips (2.0-3.0 mm in length) were excised from 4-week-old stock shoots and stepwise precultured with increased sucrose concentrations from 0.3 to 0.5 M, each concentration for 1 day. The precultured shoot tips were then loaded for 20 min with a solution composed of 2 M glycerol and 0.5 M sucrose, before exposure to PVS3 for 3 h at room temperature. Dehydrated shoot tips were transferred onto aluminum foils (2×0.8 cm), prior to direct immersion into liquid nitrogen (LN) for cryostorage. For thawing, frozen aluminum foils were moved from LN and immediately transferred into unloading solution composed of liquid MS containing 1.2 M sucrose. After incubation at room temperature for 20 min, shoot tips were post-cultured on solidified MS medium containing 0.3 M sucrose for 2 days and then transferred onto a recovery medium for shoot regrowth. With this procedure, 94% shoot tips survived, and 58% shoot tips regenerated into shoots following cryopreservation.

Abstract

Potato (Solanum tuberosum L.) is one of the most important crops grown in Norway, and virus-free plants are required for commercial potato production and for preservation of potato germplasm. The present study evaluates three in vitro therapies – meristem culture, cryotherapy, and chemotherapy combined with thermotherapy – to eliminate viruses from eight historically valuable potato cultivars belonging to the Norwegian potato germplasm. Potato virus Y, potato virus M, potato virus X and potato virus S were present in eight selected old potato cultivars due to long-term conservation in open field. Double-antibody sandwich enzyme-linked immunological assay (DAS-ELISA) and biological indicators were the standard tests used to confirm virus infection in our study. Six virus-free plants from four potato cultivars were obtained after meristem culture, and no virus-free potato cultivars were obtained after cryotherapy. Virus-free frequency for eight different potato cultivars after combining chemotherapy with thermotherapy varied from 36.4% to 100%, with single virus elimination rates of between 74.2% and 92.9%. Chemotherapy compared with thermotherapy was the most effective of the three in vitro therapies used in this study. Highly sensitive small RNA high-throughput sequencing (HTS) was used to evaluate the virus status of potato virus-free materials after virus eradication, and no virus was found, which was consistent with the results of DAS-ELISA and biological indicators. Small RNA HTS has been reported for the first time to evaluate the virus status after virus elimination and to control virus-free potato nuclear stocks.

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Abstract

Virus diseases have been a great threat to production of economically important crops. In practice, the use of virus-free planting material is an effective strategy to control viral diseases. Cryotherapy, developed based on cryopreservation, is a novel plant biotechnology tool for virus eradication. Comparing to the traditional meristem culture for virus elimination, cryotherapy resulted in high efficiency of pathogen eradication. In general, cryotherapy includes seven major steps: (1) introduction of infected plant materials into in vitro cultures, (2) shoot tip excision, (3) tolerance induction of explants to dehydration and subsequent freezing in liquid nitrogen (LN), (4) a short-time treatment of explants in LN, (5) warming and post-culture for regeneration, (6) re-establishment of regenerated plants in greenhouse conditions, and (7) virus indexing.

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Abstract

Viral diseases (a biotic stress) and salinity (an abiotic stress) have been/are the two major constraints for sustainable development of the world’s agricultural production including potato. Crops grown in field are often exposed simultaneously to abiotic and biotic stress, and responses of plants to co-stress by two or more factors may differ from those to each of the multiple stresses. Using in vitro cultures, we demonstrated that virus infection (singly and in combination) or salt, and co-stress by virus infection (singly and in combination) and salt significantly reduced growth and microtuber production, and caused severely oxidative cell damage determined by levels of O2·− and methane dicarboxylic aldehyde, and H2O2 localization in situ. Alterations in physiological metabolism by increasing total soluble sugar and free proline, and by decreasing chlorophyll content are responses of potato plantlets to virus infection (singly and in combination) or salt stress and co-stress by virus infection (singly and in combination) and salt. Oxidative cell damage and reduced chlorophyll content caused by virus and/or salt are believed to be responsible for the reduced growth, eventually resulting in decreased tuber yield. Results reported here would help us to better understand possible mechanism of reduced tuber yield by virus infection and/or salt stress.

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Abstract

Availability of and easy access to diverse plant viruses and viroids is a prerequisite in applied and basic studies related to viruses and viroids. Long-term preservation of viruses and viroids is difficult. A protocol was described for long-term preservation of potato leafroll virus (PLRV), potato virus S (PVS), and potato spindle tuber viroid (PSTVd) in cryopreserved shoot tips of potato cv. Zihuabai. Shoot regrowth levels following cryopreservation were higher in 1.5 mm-shoot tips (58–60%) than in 0.5-mm-ones (30–38%). All shoots recovered from 0.5-mm-shoot tips were PVS- and PSTVd-preserved, but none of them were PLRVpreserved. Cryopreservation of 1.5-mm-shoot tips resulted in 35% and 100% of PLRV- and PVS- and PSTVd-preserved shoots. Studies on cell survival patterns and virus localization provided explanations to the varying PLRV-preservation frequencies produced by cryopreservation of the two sizes of shoot tips. Although micropropagation efficiencies were low after 12 weeks of subculture following cryopreservation, similar efficiencies were obtained after 16 weeks of subculture in pathogen-preserved shoots recovered from cryopreservation, compared with the diseased in vitro stock shoots (the control). Pathogen concentrations in the three pathogens-preserved shoots analyzed by qRT-PCR were similar to those in micropropagated shoots. The three pathogens cryopreserved in shoot tips were readily transmitted by grafting and mechanical inoculation to potato plants. PLRV, PVS, and PSTVd represent a diverse range of plant viruses and viroid in terms of taxonomy and infectious ability. Therefore, shoot tip cryopreservation opens a new avenue for long-term preservation of the virus and viroid.

Abstract

Chrysanthemum stunt viroid (CSVd) was first reported in US in the 1940s and is widespread in the world wherever chrysanthemum is grown. Cryotherapy of shoot tips, a new biotechnology developed in the recent years, is a novel application of plant cryopreservation techniques that allows pathogen eradication at a high frequency. Existing studies have proven that this technique can efficiently eradicate pathogens such as virus, phytoplasma and bacterium. However, up to now, there has been no report on viroid eradication. In the present study, we attempted to establish a droplet vitrification cryotherapy method for Argyranthemum and to apply it to eradicate CSVd. Results obtained so far demonstrated that cryotherapy of shoot tips alone failed to eradicate CSVd from the infected shoot tips of Argyranthemum maderense ‘Yellow Empire’. Using in situ hybridization of CSVd and histological analysis, we found that CSVd can invade meristematic cells and at the same time, these cells were able to survive following cryotherapy. These findings explained why cryotherapy of shoot tips alone could not be efficient enough to eradicate CSVd from the diseased materials. Further studies combining cold treatment with cryotherapy are under investigation for CSVd eradication.