Qiaochun Wang

Combined thermotherapy and cryotherapy for efficient virus eradication: relation of virus distribution, subcellular changes, cell survival and viral RNA degradation in shoot tips.

Accumulation of viruses in vegetatively propagated plants causes heavy yield losses. Therefore, supply of virus-free planting materials is pivotal to sustainable crop production. In previous studies, Raspberry bushy dwarf virus (RBDV) was difficult to eradicate from raspberry (Rubus idaeus) using the conventional means of meristem tip culture. As shown in the present study, it was probably because this pollen-transmitted virus efficiently invades leaf primordia and all meristematic tissues except the least differentiated cells of the apical dome. Subjecting plants to thermotherapy prior to meristem tip culture heavily reduced viral RNA2, RNA3 and the coat protein in the shoot tips, but no virus-free plants were obtained. Therefore, a novel method including thermotherapy followed by cryotherapy was developed for efficient virus eradication. Heat treatment caused subcellular alterations such as enlargement of vacuoles in the more developed, virus-infected cells, which were largely eliminated following subsequent cryotherapy. Using this protocol, 20-36% of the treated shoot tips survived, 30-40% regenerated and up to 35% of the regenerated plants were virus-free, as tested by ELISA and reverse transcription loop-mediated isothermal amplification. Novel cellular and molecular insights into RBDV-host interactions and the factors influencing virus eradication were obtained, including invasion of shoot tips and meristematic tissues by RBDV, enhanced viral RNA degradation and increased sensitivity to freezing caused by thermotherapy, and subcellular changes and subsequent death of cells caused by cryotherapy. This novel procedure should be helpful with many virus-host combinations in which virus eradication by conventional means has proven difficult.

Elimination of grapevine virus A (GVA) by cryopreservation of in vitro-grown shoot tips of Vitis vinifera L

Abstract Methods for the cryopreservation of in vitro-grown shoot tips of grapevine were recently developed [1] , [2] . The present study demonstrates that grapevine virus A (GVA) can be successfully eliminated from naturally infected grapevine by cryopreservation of in vitro-grown shoot tips. The various steps taken before freezing in liquid nitrogen did not, by themselves, eliminate GVA. However, the freezing step resulted in 97% GVA elimination. The size of the shoot tips used for cryopreservation influenced their survival rate, while viral eradication was independent of their size in the range of 0.5–2.0 mm. In comparison, plant regeneration from meristems increased with size, and meristems of 0.1 mm completely failed to regenerate. Regeneration from 0.4-mm meristems reached 100%, but none of the regenerated plantlets were GVA-free. Meristems of 0.2 mm resulted in only 12% GVA-free plants. Frequency of GVA elimination was not affected by the cryopreservation procedure, be it encapsulation–dehydration or vitrification. Leaf morphology of plants regenerated from cryopreserved shoot tips was similar to that from control shoot tips. Results from the present study suggest cryopreservation of shoot tips as a simple and efficient method for eliminating GVA from infected grapevine plants.

Cryopreservation of in vitro-grown shoot tips of raspberry (Rubus idaeus L.) by encapsulation–vitrification and encapsulation–dehydration

The first efficient cryopreservation procedure for in vitro-grown shoot tips of raspberry (Rubus idaeus L.) has been developed based on encapsulation–vitrification (EnVi) and encapsulation–dehydration (EnDe). EnVi resulted in higher survival (85%) and regrowth (75%) of cryopreserved shoot tips than EnDe (65 and 50%, respectively). In both cryogenic procedures, shoots regenerated from cryopreserved shoot tips without intermediary callus formation. Histological studies showed that a much larger number of meristematic cells survived following EnVi than EnDe. The EnVi procedure was applied to seven raspberry genotypes with an average survival and regrowth of 71 and 68%, respectively. Regenerated plants showed normal morphology. Results here indicate EnVi as a simple and efficient method for long-term preservation of R. idaeus germplasm.

Cryopreservation of grapevine (Vitis vinifera L.) embryogenic cell suspensions by encapsulation-dehydration and subsequent plant regeneration

Embryogenic cell suspensions of grapevine (Vitis vinifera L.) were successfully cryopreserved by encapsulation-dehydration and subsequently regenerated into plants. Embryogenic cell suspensions were first-step precultured for 2 days in MGN liquid medium supplemented with increasing sucrose concentrations of 0.25, 0.5, 0.75 and 1 M. Precultured cells were then encapsulated and second-step precultured in MGN liquid medium containing 1 M sucrose for several days. Following preculture, encapsulated cells were dehydrated prior to direct immersion in liquid nitrogen for 1 h. After thawing, cryopreserved cells were post-cultured on MGN medium for survival. Surviving cells were then transferred to different media for regrowth and plant regeneration, respectively. An optimal viability of cryopreserved cells was achieved when encapsulated cells were dehydrated to 20.6% water content and subjected to 2–4 days of preculture on 1 M sucrose. MGN solid medium containing 2.5 g/l AC was found to promote viability of cryopreserved cells. Although cryopreserved cells showed a 5-day lag phase in regrowth, and their increase in fresh weight was only half that of control cells, their growth pattern was the same as that of control cells after two subcultures, following re-establishment in cell suspensions. Cryopreservation promoted embryogenesis and subsequent plant germination, compared with both the control and dehydration treatments. Leaf morphologies of plants regenerated from cryopreserved cells appeared to be similar to those of plants from control cells.

Elimination of two viruses which interact synergistically from sweetpotato by shoot tip culture and cryotherapy

Sweet potato chlorotic stunt virus (SPCSV; Closteroviridae) and Sweet potato feathery mottle virus (SPFMV; Potyviridae) interact synergistically and cause severe diseases in co-infected sweetpotato plants (Ipomoea batatas). Sweetpotato is propagated vegetatively and virus-free planting materials are pivotal for sustainable production. Using cryotherapy, SPCSV and SPCSV were eliminated from all treated single-virus-infected and co-infected shoot tips irrespective of size (0.5-1.5mm including 2-4 leaf primordia). While shoot tip culture also eliminated SPCSV, elimination of SPFMV failed in 90-93% of the largest shoot tips (1.5mm) using this technique. Virus distribution to different leaf primordia and tissues within leaf primordia in the shoot apex and petioles was not altered by co-infection of the viruses in the fully virus-susceptible sweetpotato genotype used. SPFMV was immunolocalized to all types of tissues and up to the fourth-youngest leaf primordium. In contrast, SPCSV was detected only in the phloem and up to the fifth leaf primordium. Because only cells in the apical dome of the meristem and the two first leaf primordia survived cryotherapy, all data taken together could explain the results of virus elimination. The simple and efficient cryotherapy protocol developed for virus elimination can also be used for preparation of sweetpotato materials for long-term preservation.

Cryopreservation of grapevine (Vitis spp.) embryogenic cell suspensions by encapsulation-vitrification

Embryogenic cell suspensions of two grapevine rootstocks: 110 Ritcher (V. berlandieri × V. rupestris), 41B (V. vinifera × V. berlandieri) and several table grape and wine cultivars (Vitis vinifera) were successfully cryopreserved by the encapsulation–vitrification method. Embryogenic cell suspensions were precultured for 3 days in liquid MGN medium supplemented with daily increasing sucrose concentrations of 0.25, 0.5, 0.75 M. Precultured cells were encapsulated and directly dehydrated with a highly concentrated vitrification solution prior to immersion in liquid nitrogen for 1 h. After rewarming at 40 °C for 3 min, cryopreserved cells were post-cultured on solid MGN medium supplemented with 2.5 g l−1 activated charcoal. Surviving cells were transferred to solid MGN medium for regrowth or solid MG medium for embryo development and then to solid WPM for plant regeneration. Optimal viability was 42–76% of cryopreserved cells when cell suspensions were precultured with a final sucrose concentration of 0.75 M and dehydrated with PVS2 at 0 °C for 270 min. Biochemical analysis showed that sucrose preculture caused changes in levels of total soluble protein and sugars in cell suspensions. Although the increase in fresh weight was significantly lower in cryopreserved cells than in control cells, the growth pattern of the cryopreserved cells and control cells was the same after two subcultures, following re-establishment in cell suspensions. Protocol developed in this study suggests a universal and highly efficient cryopreservation system suitable for several genetically diversed Vitis species.

Cryotherapy of shoot tips: novel pathogen eradication method

Cryotherapy is a novel application of plant cryopreservation techniques that allows pathogen eradication at a high frequency. It eliminates plant pathogens such as viruses, phytoplasmas and bacteria by briefly treating shoot tips in liquid nitrogen using cryopreservation protocols. Healthy plants are regenerated from the surviving pathogen-free meristematic tissue. The method facilitates treatment of large numbers of samples and is independent of shoot tip size. It has the potential to replace more traditional methods like meristem culture.

Cryopreservation of in vitro-grown shoot tips of grapevine by encapsulation-dehydration.

In vitro-grown shoot tips of the LN33 hybrid (Vitis L.) and cv. Superior (Vitis vinifera L.) were successfully cryopreserved by encapsulation-dehydration. Encapsulated shoot tips were precultured stepwise on half-strength MS medium supplemented with increasing sucrose concentrations of 0.25, 0.5, 0.75 and 1.0 M for 4 days, with one day for each step. Following preculture, encapsulated shoot tips were dehydrated prior to direct immersion in liquid nitrogen for 1 h. After thawing, cryopreserved shoot tips were post-cultured on a post-culture medium for survival. An optimal survival of cryopreserved shoot tips was achieved when encapsulated shoot tips were dehydrated to 15.6 and 17.6% water content for the LN33 hybrid and cv. Superior, respectively. Comparison between the effects of dehydration with silica gel and by air drying on cryopreserved shoot tips, showed that survival was dependent on water content, not on dehydration method. The thawing method markedly affected survival of cryopreserved shoot tips, and thawing at 40 °C for 3 min was found best. No callus formation and fastest shoot elongation were obtained when cryopreserved shoot tips were post-cultured on the post-culture medium composed of half-strength MS supplemented with 1 mg l−1 BA and 0.1 mg l−1 NAA. With these optimized parameters, 60 and 40% survival of cryopreserved shoot tips were obtained for the LN33 hybrid and cv. Superior, respectively.

Localization of fruit tree viruses by immuno-tissue printing in infected shoots of Malus sp. and Prunus sp.

Immuno-tissue printing protocols for the localization of apple chlorotic leaf spot virus (ACLSV), stem grooving virus (SGV) and plum pox virus (PPV) in shoots of Prunus and Malus in vitro have been established for routine diagnosis in a virus elimination program. Since these viruses belong to different virus genera, the protocols were adapted according to the properties of the virus under investigation. Accumulation of ACLSV was highest in the base of the stem and decreased towards the apex of the shoots. ACLSV was found in the epidermis, the cortex, in the vascular bundles, but seldom in the pith tissue of in vitro apple shoots. ACLSV immuno-tissue printing was as sensitive as ELISA and the intensity of color signals in immuno-tissue prints correlated with absorbance values by two-step ELISA. SGV could be detected by immuno-tissue prints at infectivity levels, where it reacted negative in ELISA. SGV accumulated in the vascular bundles, occurred locally in the parenchymatic tissue, was found in high amounts in young leaves near the meristem, but not within the meristem. PPV was detected in all tissue types of stem sections with an irregular pattern reflecting the in vivo situation causing problems with detection. Discrimination of poorly and heavily infected shoots was possible with the naked eye.

Sweetpotato in China

The pattern of grain utilization in China has changed significantly in the past decades, a trend that is likely to continue in the future 1. As incomes increased, rice and wheat as staple food slowly took the place of sweetpotato, maize and other course grains, which in turn have increasingly been used as animal feed. However, despite the significant shift in the use of both maize and sweetpotato from food to feed, Chinas domestic feed supply is expected to lag behind the rapidly increasing demand for livestock meat products brought about by the expansion of consumption as a function of increased income. Trade liberalization and China?s membership in the World Trade Organization (WTO) are expected to exacerbate the deficit of feed grains, particularly maize. Whether China could or would be willing to import a large amount of maize is an issue of rising concern. Alternative solutions, especially substitution of other domestically produced feed such as sweetpotato are of importance, politically and economically.