ABSTRACT
Vanilla planifolia Jacks. ex Andrews is the vanilla species with the most commercial and greatest economic importance. It has been used as a case study in different cryopreservation studies that involve three vitrification-based approaches: droplet-vitrification (D-V), V-cryoplate (V-Cp) and D-cryoplate (D-Cp). The aim of this study was to compare the impact of these cryogenic techniques on vegetative growth (survival, stem length and leaf number) between cryo-derived plants and in vitro-derived controls during 12 months of greenhouse growth. Genetic stability was also assessed using the inter-simple sequence repeat (ISSR) markers. There were no significant differences found in the survival and stem lengths of the in vitro-derived regenerants and cryo-derived plants. A significant increase in the number of leaves was only detected in cryo-derived plants when using the V-Cp method. The electrophoretic profiles, based on seven ISSR primers, detected low variability: 81 total bands and 27% polymorphism. This is the first report on the assessment of vegetative growth and genetic integrity in cryo-derived V. planifolia plants recovered under greenhouse conditions. Of the three cryogenic approaches, D-Cp appears to yield V. planifolia regenerants plants with more vigorous vegetative growth and a lower level of polymorphism. Future research should focus on the reproductive growth of vanilla regenerants.
ABSTRACT
Agave tequilana Weber cultivar 'Chato' represents an important genetic supply of wild severely in decline populations of 'Chato' for breeding and transformation programs. In this work, the indirect somatic embryogenesis and cryopreservation of Somatic Embryos (SEs) were investigated using the 'Chato' cultivar as a study case. METHODS: Embryogenic calli were induced by the cultivation of 1 cm of young leaves from in vitro plants on MS semisolid medium supplemented with 24.84, 33.13, 41.41, 49.69, and 57.98 µM 4-amino-3,5,6-trichloro-2- pyridinecarboxylic acid (picloram) in combination with 2.21, 3.32, and 4.43 µM 6-benzylaminopurine (BAP). The origin and structure of formed SEs were verified by histological analysis. Cryopreservation studies of SEs were performed following the V-cryoplate technique and using for dehydration two vitrification solutions (PVS2 and PVS3). RESULTS: The highest average (52.43 ± 5.74) of produced SEs and the Embryo Forming Capacity (estimated index 52.43) were obtained using 49.69 µM picloram and 3.32 µM BAP in the culture medium. The highest post-cryopreservation regrowth (83%) and plant conversion rate (around 70%) were achieved with PVS2 at 0 °C for 15 min. CONCLUSION: Our work provides new advances about somatic embryogenesis in Agave and reports the first results on cryopreservation of SEs of this species.
ABSTRACT
In this work, we investigated the effect of different osmoprotective treatments and of cryopreservation using a droplet-vitrification (D-V) protocol to eliminate sugarcane mosaic virus (SCMV) of shoot-tips excised from in vitro propagated infected plantlets. Shoot-tips of sugarcane (Saccharum spp. L.) were precultured on semisolid MS medium supplemented with 0.3 M sucrose for 1 day, loaded in solution with 0.4 M sucrose and 2 M glycerol for 30 min and exposed to plant vitrification solution 2 for 15 min at room temperature prior to ultra-rapid cooling in liquid nitrogen. Virus indexing was performed by the DAS-ELISA immunoenzymatic test. The presence of SCMV was confirmed in the donor-plantlets derived of infected field material. No virus was detected in the regenerated plantlets from shoot-tips subjected to cryopreservation protocol. The progressive decrease in absorbances occurred from the first preculture treatment and no significant differences (P ≤ 0.05) were found with respect to following steps of D-V protocol. These results indicate that the osmotic dehydration treatments (osmotherapy) and cryopreservation (cryotherapy) may be potentially effective strategies to remove the SCMV from infected plants.
ABSTRACT
The effect of subculture cycles on somaclonal variation of V. planifolia using intersimple sequence repeat (ISSR) markers was analyzed. Nodal segments of 2 cm in length were established in vitro and multiplied by 10 subculture cycles in Murashige and Skoog (MS) medium supplemented with 8.86 µM BAP (benzylaminopurine). After 45 days in each culture, the length and number of shoots per explant were evaluated. For ISSR markers, ten shoots per each subculture and the mother plant were used. Ten ISSR primers were used and a total of 118 bands were obtained. The polymorphism (%) was calculated and a dendrogram based on Jaccard's genetic distance between the subcultures and the donor plant was obtained. These results show that the multiplication rate tends to increase until subculture five, whereas shoot length decreases as the number of subcultures increases. The ISSR markers revealed an increase in the polymorphism percentage after the fifth culture cycle. The dendrogram showed the formation of two groups. The first group, with less genetic variability, is the donor plant and subcultures 1-5; the second group has greater genetic distance and is formed by subcultures 6-10. The results revealed that the number of subcultures with 8.86 µM BAP is a factor that affects the somaclonal variation during in vitro regeneration of V. planifolia. In conclusion, the subculture number affects somaclonal variation and in vitro development of V. planifolia.
ABSTRACT
Encapsulation-dehydration is a cryopreservation technique based on the technology developed for producing synthetic seeds, i.e. the encapsulation of explants in calcium alginate beads. Encapsulated explants are then precultured in liquid medium with a high sucrose concentration and partially desiccated before freezing. Encapsulating the explants allows the subsequent application of very drastic treatments including preculture with high sucrose concentrations and desiccation to low moisture contents which would be highly damaging or lethal to non-encapsulated samples. An encapsulation-dehydration protocol comprises the following steps: pretreatment, encapsulation, preculture, desiccation, freezing and storage, thawing and regrowth. Encapsulation-dehydration has been applied to around 40 different plant species. The optimization of the successive steps of the encapsulation-dehydration protocol is illustrated for sugarcane apices.
