Cryopreservation of Hazelnut (Corylus avellana L.) Axillary Buds from In Vitro Shoots Using the Droplet Vitrification Method

Cryopreservation by droplet vitrification was applied to hazelnut (Corylus avellana L.). axillary buds of the Italian cultivated variety Tonda Gentile Romana, which were collected from in vitro growing shoots, immersed in ice cooled PVS2 or PVS3 for 60 or 90 min, then transferred to a droplet of vitrification solution, placed on a strip of aluminium foil, and plunged into liquid nitrogen (LN). Additionally, the effect on the recovery of the mother plant after cryopreservation was evaluated, following a cold pre-treatment at 4 °C for 3 months. The highest regrowth percentage (56.7%) was obtained after applying PVS3 for 60 min, while the application of PVS2 for the same amount of time reduced regrowth to 41.5%. Increasing the exposure to vitrification solutions to 90 min reduced regrowth to 43.3% when PVS3 was applied, and 35.6% if PVS2 was used. The cold pre-treatment on the mother plant did not significantly improve overall regrowth. The cryopreservation process did not decline the rooting ability of the recovered shoots.

Optimization of a Cryopreservation Method for the Endangered Korean Species Pogostemon yatabeanus Using a Systematic Approach: The Key Role of Ammonium and Growth Regulators

Cryopreservation provides a secure long-term conservation option for rare and endangered plant species with non-orthodox or limitedly available seeds. Wide application of cryopreservation to biobank wild flora is hampered by the need to re-optimize nearly all protocol steps for every new species. We applied a systematic approach to simplify optimization of a multi-stage droplet-vitrification method for the endangered wetland Korean species, Pogostemon yatabeanus. This approach consisted of a standard procedure pre-selected based on material type and size, which was complemented with 11 additional treatments to reveal the most impactful conditions. Effect of ammonium nitrate at various protocol steps was also tested. The highest shoot tip survival (92%) and plant regeneration (90%) after cryopreservation were achieved using preculture with 10% sucrose followed by 40 min osmoprotection and 60 min treatment with vitrification solution A3-80% (33.3% glycerol + 13.3% dimethyl sulfoxide + 13.3% ethylene glycol + 20.1% sucrose) on ice. A three-step regrowth procedure starting with ammonium-free medium with 1 mg/L GA3 and 1 mg/L BA followed by ammonium-containing medium with and without growth regulators was essential for the development of healthy plants from cryopreserved shoot tips. This approach enables fast optimization of the cryopreservation procedure for new osmotic stress-sensitive plant species.

Improved Conservation of Coffee (Coffea arabica L.) Germplasm via Micropropagation and Cryopreservation

Coffee (Coffea spp.) is an important tropical agricultural crop that has significant economic and social importance in the world. The ex situ conservation of plant genetic resources through seeds is not feasible due to the sensitivity of coffee seed to desiccation and low temperatures. The cryopreservation of zygotic embryos may allow for an efficient and long-term storage of coffee germplasm. This study describes the cryopreservation methods for conserving zygotic embryos of Coffea arabica L. for the long-term conservation of currently available germplasm. Zygotic embryos were successfully cryopreserved in liquid nitrogen at −196 °C under controlled environmental conditions with either droplet-vitrification or encapsulation–vitrification protocols without dehydration. Zygotic embryos had the highest regrowth (100%) following droplet-vitrification cryopreservation using the Plant Vitrification Solution 3 (PVS3) for 40 min at 23 °C. In the case of encapsulation–vitrification using PVS3 for 40 min at 23 °C, the embryo regeneration response was 78%. Plantlets were recovered following shoot multiplication using a temporary immersion system (TIS) and in vitro rooting. The prolific rooting of shoots was observed after 4 weeks of culture in the liquid medium with plugs made of the inert substrate Oasis® In vitro Express (IVE) compared to the semi-solid medium. The successful cryopreservation of coffee zygotic embryos using droplet vitrification and encapsulation–vitrification followed by micropropagation in temporary immersion culture system has not been reported earlier and together these technologies are anticipated to further facilitate the initiatives for the conservation and distribution of coffee germplasm.

Comparative Study of Three Vitrification Solutions for an Effective Droplet-vitrification Cryopreservation Procedure for Pfaffia Glomerata (Spreng.) Pedersen

Aims: The objective of this research was to establish a cryopreservation protocol for shoot tips (ST) of in vitro P. glomerata using the droplet-vitrification technique. Study Design: The experimental design was a factorial, with four factors, arranged in a completely randomized design. Three vitrification solutions (PVS2, PVS3, PVS4), three times (20, 40, 60 min) and two temperatures (25 ± 2 °C and 0 °C) of treatment with the solutions, followed by freezing (LN+) or not (LN-) with liquid nitrogen (LN) were tested. All tests were performed using six replicates and the results analysed using Two-way ANOVA and Tukey’s tests and expressed as the mean ± the standard error of the means (SEM) deviation. Place and Duration of Study: Laboratory of Plant Cryobiology, Embrapa Genetic Resources and Biotechnology, over a two-year period. Methodology: ST excised from in vitro plantlets were pre-cultured overnight (19h), treated with a loading solution (LS) and three different vitrification solutions (PVS2, PVS3, PVS4) prior to freezing in LN. Treatment with the vitrification solutions was carried out at 0 or 25°C, for 20, 40 or 60 min. For freezing, drops of the vitrification solutions containing a single ST were dispensed on aluminum foil strips and the strips were submerged in LN (-196°C). For thawing, foil strips were submerged into unloading solution (US) at 40 ± 2°C, for three min. Thawed ST were transferred to regeneration medium and cultured in vitro. Results: Highest regeneration percentages after cryopreservation were 82% for ST treated with PVS3, at 0°C, for 60 min; 32% for ST treated with PVS4 at 25°C for 60 min or 0°C for 40 min and 22% for those treated with PVS2 at 0°C for 60 min. Conclusion: Droplet-vitrification is a suitable technique to ensure survival of P. glomerata ST after cryopreservation. This procedure can be applied to establish germplasm collections of this medicinal species in gene banks.