Cannabis has developed into a multi-billion dollar industry that relies on clonal propagation of elite genetics with desirable agronomic and chemical phenotypes. While the goal of clonal propagation is to produce genetically uniform plants, somatic mutations can accumulate during growth and compromise long-term genetic fidelity. Cryopreservation is a process in which tissues are stored at cryogenic temperatures, halting cell division and metabolic processes to facilitate high fidelity germplasm preservation. In this study, a series of experiments were conducted to optimize various stages of cryopreservation and develop a protocol for long-term germplasm storage of Cannabis sativa. The resulting protocol uses a standard vitrification procedure to cryopreserve nodal explants from in vitro shoots as follows: Nodes were cultured for 17 hours in a pre-culture solution (PCS), followed by a 20 minute treatment in a loading solution (LS), and a 60 minute incubation in plant vitrification solution 2 (PVS2). The nodes were then flash frozen in liquid nitrogen, re-warmed in an unloading solution at 40°C, and cultured on basal MS culture medium in the dark for 5 days followed by transfer to standard culture conditions. This protocol was tested across 13 genotypes to assess the genotypic variability. The protocol was successful across all 13 genotypes, but significant variation was observed in tissue survival (43.3-80%) and regrowth of shoots (26.7-66.7%). Plants grown from cryopreserved samples were morphologically and chemically similar to control plants for most major traits, but some differences were observed in the minor cannabinoid and terpene profiles. While further improvements are likely possible, this study provides a functional cryopreservation system that works across multiple commercial genotypes for long-term germplasm preservation.

Recent developments in the cryopreservation space has increased the trend in germplasm collections established through cryopreserved in vitro material. Cryopreservation of recalcitrant seeds through embryos and embryonic axes, is not uncommon. Tropical and sub-tropical plants are not acclimated to the cold season, therefore have no in-built natural resilience to the cold. Also, larger seeds from trees, such as avocado (Persea americana Mill.), mango (Mangifera indica) and durian (Durio zibethinus L.) are sensitive to desiccation, chilling and freezing stress, making them unsuitable for seed banking or cryopreservation. Alternatively, as seeds do not carry the same genetic make-up as the mother plant, especially in the context of woody rainforest species of which the cross-pollination is dominant; seed conservation does not serve the purpose of germplasm preservation. Other plant material and methods are needed for these plants to be successfully stored in liquid nitrogen (LN). One such method commonly used is shoot-tip cryopreservation which ensures the clonal fidelity of germplasm. There are many problems when using shoot tips of tropical recalcitrant-seeded species. These include: 1) the toxic effects of cryoprotective agents towards structural integrity; 2) optimum developmental stage for success and 3) oxidative stress associated with excision injury leading to necrosis triggering cell death and hindering regeneration for the shoot tips in culture. A pre-requisite for any cryopreservation system is the availability of an established tissue culture regeneration platform. This review will outline conservation strategies for avocado with special emphasis on attempts and improvements made in the cryopreservation space for storing this horticulturally important crop ‘avocado’ at ultra-low temperatures.

The liquid droplet cooling technique for fast scanning chip calorimetry (FSC) is introduced, increasing the cooling rate for large samples on a given sensor. Reaching higher cooling rates and using a gas as the cooling medium, the common standard for ultra-fast temperature control in cooling, requires reducing the lateral dimensions of the sample and sensor. The maximum cooling rate is limited by the heat capacity of the sample and the heat exchange between the gas and the sample. The enhanced cooling performance of the new liquid droplet cooling technique is demonstrated for both metals and polymers, on examples of solidification of large samples of indium, high-density polyethylene (HDPE), and poly (butylene 2,6-naphthalate) (PBN). It was found that the maximum cooling rate can be increased up to 5 MK/s in room temperature environment, that is, by 2 orders of magnitude, compared to standard gas cooling. Furthermore, modifying the droplet size and using coolants at different temperatures provide options to adjust the cooling rate in the temperature ranges of interest.

International guidelines are published in oncology to provide standardized information and fertility preservation (FP) care for adults and children with cancer. For benign gynecologic diseases (BGD), many recommendations are based on data coming from oncofertility studies rather than studies with a stronger and broader evidence base. The purpose of the study was to conduct a modified Delphi process for generating FP guidelines for BGD. A steering committee identified 42 potential FP practices for BGD. Then 114 key stakeholders were asked to participate in a modified Delphi process via two online survey rounds and a final meeting. Consensus was reached for 28 items. Among them, stakeholders rated age-specific information concerning the risk of diminished ovarian reserve after surgery as important but rejected proposals setting various upper and lower age limits for FP. All women should be informed about the benefit/risk balance of oocyte vitrification, in particular about the likelihood of live birth according to age. FP should not be offered in rASRM stages I and II endometriosis without endometriomas. These guidelines could be useful for gynecologists to identify situations at risk of infertility and to better inform women with BGDs who might need personalized counseling for FP.