Cryopreservation of 13 Commercial Cannabis sativa Genotypes Using In Vitro Nodal Explants

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 h in a pre-culture solution (PCS), followed by a 20-min treatment in a loading solution (LS), and a 60 min 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.

Flower Power: Floral reversion as a viable alternative to nodal micropropagation in Cannabis sativa

The legalization of Cannabis sativa L. for recreational and medical purposes has been gaining global momentum, leading to a rise in interest in Cannabis tissue culture as growers look for large-scale solutions to germplasm storage and clean plant propagation. Mother plants used in commercial propagation are susceptible to insect pests and disease and require considerable space. While micropropagation can produce disease free starting material in less space, current published in vitro micropropagation methods are not robust and few report high multiplication rates. Further, these micropropagation methods rely on photoperiod-sensitive plants which can be maintained in a perpetual vegetative state. Current methods are not adaptable to long-term tissue culture of day-neutral cultivars, which cannot be maintained in perpetual vegetative growth. In this study, we chose to develop a micropropagation system which uses C. sativa inflorescences as starting materials. This study used two cannabis cultivars, two plant growth regulators (PGR; 6-benzylaminopurine and meta-topolin) at different concentrations, and two different numbers of florets. Here we show that floral reversion occurs from meristematic tissue in C. sativa florets and that it can be used to enhance multiplication rates compared to existing in vitro methods. Floret number was shown to have a significant impact on percent reversion, with pairs of florets reverting more frequently and producing healthier explants than single florets, while cultivar and PGR had no significant effect on percent reversion. Compared with our previously published nodal culture studies, the current floral reversion method produced up to eight times more explants per tissue culture cycle. Floral reversion provides a foundation for effective inflorescence-based micropropagation systems in C. sativa.

Instant Insights: Sweetpotato

This specially formulated collection features 3 reviews of current topics and key research in sweetpotato. The first chapter examines the origin and dispersal of sweetpotato, considers in vitro germplasm storage in sweetpotato genebanks, and looks at the importance of managing sweetpotato crop wild relatives (CWR). The chapter also considers the specific issues associated with sweetpotato germplasm, as well as the application of next-generation sequencing to sweetpotato and its CWR. The second chapter reviews the development and application of genetic transformation and trait improvement to sweetpotato, including the development of sweetpotato plants which are resistant to disease and abiotic stress, and sweetpotatoes with improved starch quality and higher anthocyanin content. The final chapter examines the nutritional contribution made by OFSP (orange-fleshed sweetpotato) in poor rural communities in Malawi, Ghana, Nigeria and Burkina Faso; sustainable breeding and seed systems; and effective commercialisation and marketing to benefit the communities concerned. This chapter includes detailed case studies from Ghana and Malawi.

DKW basal salts improve micropropagation and callogenesis compared to MS basal salts in multiple commercial cultivars of Cannabis sativa

Micropropagation of Cannabis sativa is an emerging area for germplasm storage and large-scale production of clean plants. Existing protocols use a limited number of genotypes and are often not reproducible. Previous studies reported MS + 0.5 μM TDZ to be optimal for Cannabis nodal micropropagation, yet our preliminary studies using nodal explants suggested this media may not be optimal. It resulted in excessive callus formation, hyperhydricity, low multiplication rates, and high mortality rates. Following an initial screen of four commonly used basal salt mixtures (MS, B5, BABI, and DKW), we determined that DKW produced the healthiest plants. In a second experiment, the multiplication rate and canopy area of explants grown on MS + 0.5 μM TDZ and DKW + 0.5 μM TDZ were compared using five drug-type cultivars to determine if the preference for DKW was genotype-dependent. Four cultivars had significantly higher multiplication rates on DKW + 0.5 μM TDZ with the combined average being 1.5x higher than explants grown on MS + 0.5 μM TDZ. The canopy area was also significantly larger on DKW + 0.5 μM TDZ for four cultivars with the combined average being twice as large as the explants grown on MS + 0.5 μM TDZ. In the third experiment, callogenesis was compared using a range of 2,4-D concentrations (0-30 μM) on both MS and DKW and similarly, callus growth was superior on DKW. This study presents the largest comparison of basal salt compositions on the micropropagation of five commercially grown Cannabis cultivars to date.

Propagation of Cynometra beddomei Prain (Leguminose) through clones and seeds

Detailed studied were conducted on the efficacy of clonal and seed germination behaviour of an endemic leguminous tree (C.b.) in Western Ghats. The study revealed maximum stem rooting (75%) and air layering success (60%) by the use of IBA 3000 and IBA 1000ppm respectively. The moderately recalcitrant type of seeds with 51% moisture content had shown 100% germination and 40% when attained critical moisture content at 38 to 40%. Overnight water soaking influenced early germination (5-6 days) than control sets. The present study is therefore first time optimize clonal and seed propagation methods of the species further to raise clonal genetic stock and germplasm storage for effective ex situ conservation from its local extinction.

Sweetpotato genetic resources: today and tomorrow

The sweetpotato (Ipomoea batatas (L.) Lam.) is the sixth most important food crop on a global scale. While China accounts for about 80% of global production, Nigeria, Uganda, Indonesia and Tanzania are also large producers of sweetpotato. The chapter examines the origin and dispersal of sweetpotato, including archaeological data for the early distribution of the crop, before moving on to considering its general botany. The chapter considers in vitro germplasm storage in sweetpotato genebanks, as well as issues of quality control. The chapter looks at the importance of managing sweetpotato crop wild relatives (CWR) and examines plant quarantine and phytosanitary issues and the status of genebanks under international treaties. The chapter considers a number of specific issues associated with sweetpotato germplasm. Finally, the chapter looks at the application of next-generation sequencing to sweetpotato and its CWR, before looking ahead to future trends in this area.

Breakthroughs and new horizons in reproductive biology of rare and endangered animal species

Because of higher extinction rates due to human and natural factors, more basic and applied research in reproductive biology is required to preserve wild species and design proper strategies leading to sustainable populations. The objective of the review is to highlight recent, inspiring breakthroughs in wildlife reproduction science that will set directions for future research and lead to more successes in conservation biology. Despite new tools and approaches allowing a better and faster understanding of key mechanisms, we still know little about reproduction in endangered species. Recently, the most striking advances have been obtained in nonmammalian species (fish, birds, amphibians, or corals) with the development of alternative solutions to preserve fertility or new information about parental nutritional influence on embryo development. A novel way has also been explored to consider the impact of environmental changes on reproduction—the allostatic load—in a vast array of species (from primates to fish). On the horizon, genomic tools are expected to considerably change the way we study wildlife reproduction and develop a concept of “precision conservation breeding.” When basic studies in organismal physiology are conducted in parallel, new approaches using stem cells to create artificial gametes and gonads, innovations in germplasm storage, and more research on reproductive microbiomes will help to make a difference. Lastly, multiple challenges (for instance, poor integration of new tools in conservation programs, limited access to study animals, or few publication options) will have to be addressed if we want reproductive biology to positively impact conservation of biodiversity.

The preservation of somatic embryos of papaya derived from papaya lateral shoots after being stored in cryopreservation to maintain plant genetic information in the future

Hervani D, Efendi D, Suhartanto MR, Purwoko BS. 2018. The preservation of somatic embryos of papaya derived from papaya lateral shoots after being stored in cryopreservation to maintain plant genetic information in the future. Biodiversitas 19: 724729. Germplasm storage of papaya is very important because this plant easily adapts to genetic changes due to environmental conditions and open system pollination, so it is necessary to retain the current genetics resources in order to conserve the genetic information. The storage of the vegetative part of the plant with cryopreservation is expected to retain the plant's genetic information in the future. Cryopreservation is the method for germplasm storage using liquid nitrogen at temperature of -196oC This experiment aimed to obtain the growth ability of papaya lateral shoots to produce somatic embryos after being stored by cryopreservation. The experiment was designed in factorial by Completely Randomized Design with two factors.The first factor was the immersion time duration in PVS2 as cryoprotectant solution with 5 treatments of immersion duration of 0, 10, 20, 30, and 40 minutes. Second factor was culture medium for cultivated the lateral shoot which was added with plant growth regulators such as BA (benzyl adenine) and NAA (naphthalene acetic acid) at levels of 0, 1, 2, 3, and 4 mg l-1, respectively. The results showed that the immersion of lateral shoot in cryoprotectants for 10 and 20 minutes gave the better plantlet survival rate after discharge from liquid nitrogen, while the treatment of culture media had not been significant difference.