Cryopreservation is considered an ideal strategy for the long-term preservation of plant genetic resources. Significant progress was achieved over the past several decades, resulting in the successful cryopreservation of the genetic resources of diverse plant species. Cryopreservation procedures often employ in vitro culture techniques and require the precise control of several steps, such as the excision of explants, preculture, osmo- and cryoprotection, dehydration, freeze-thaw cycle, unloading, and post-culture for the recovery of plants. These processes create a stressful environment and cause reactive oxygen species (ROS)-induced oxidative stress, which is detrimental to the growth and regeneration of tissues and plants from cryopreserved tissues. ROS-induced oxidative stresses were documented to induce (epi)genetic and somatic variations. Therefore, the development of true-to-type regenerants of the source germplasm is of primary concern in the application of plant cryopreservation technology. The present article provides a comprehensive assessment of epigenetic and genetic integrity, metabolic stability, and field performance of cryopreserved plants developed in the past decade. Potential areas and the directions of future research in plant cryopreservation are also proposed.

Grapevine (Vitis spp.) is one of the most economically important temperate fruit crops. Grapevine breeding programs require access to high-quality Vitis cultivars and wild species, which may be maintained within genebanks. Shoot tip cryopreservation is a valuable technique for the safe, long-term conservation of Vitis genetic resources that complements traditional field and in vitro germplasm collections. Vitis is highly susceptible to virus infections. Virus-free plants are required as propagation material for clonally propagated germplasm, and also for the global exchange of grapevine genetic resources. Shoot tip cryotherapy, a method based on cryopreservation, has proven to be effective in eradicating viruses from infected plants, including grapevine. This comprehensive review outlines/documents the advances in Vitis shoot tip cryopreservation and cryotherapy that have resulted in healthy plants with high regrowth levels across diverse Vitis species.

Lilium is one of the most popular flower crops worldwide, and some species are also used as vegetables and medicines. The availability of and easy access to diverse Lilium genetic resources are essential for plant genetic improvements. Cryopreservation is currently considered as an ideal means for the long-term preservation of plant germplasm. Over the last two decades, great efforts have been exerted in studies of Lilium cryopreservation and progress has been made in the successful cryopreservation of pollen, seeds and shoot tips in Lilium. Genes that exist in Lilium, including those that regulate flower shape, color and size, and that are resistant to cold stress and diseases caused by fungi and viruses, provide a rich source of valuable genetic resources for breeding programs to create novel cultivars required by the global floriculture and ornamental markets. Successful cryopreservation of Lilium spp. is a way to preserve these valuable genes. The present study provides updated and comprehensive information about the development of techniques that have advanced Lilium cryopreservation. Further ideas are proposed to better direct future studies on Lilium cryobiotechnology.

American chestnut (Castanea dentata), a native species of eastern North America, is an economically important deciduous hardwood tree that has been designated as endangered in Canada. The population of American chestnut trees has dwindled significantly across Southern Ontario due to chestnut blight and many of the surviving trees continue to show blight disease symptoms. American chestnut requires efficient strategies for propagation and preservation for species recovery. The objective of this study was to develop a long-term plant conservation program using micropropagation and cryopreservation protocols. An in vitro technology using a liquid-based temporary immersion system (TIS) was developed for micropropagation of American chestnut. The highest rate of shoot multiplication was observed in cultures grown in the DKW (Driver and Kuniyuki 1984) basal medium supplemented with 2.2 µM 6-benzylaminopurine and 1.0 µM gibberellic acid. More than 95% of proliferated microshoots, about 40–50 mm in size, developed roots after 30 days of culture within bioreactor vessels containing DKW basal medium supplemented with 15 µM 3-Indolebutyric acid. Rooted plantlets transplanted to the greenhouse had a survival efficiency of 82% after one month of growth. The cryopreservation protocol for germplasm preservation was developed through droplet vitrification of shoots. Optimal regeneration of shoot tips occurred from explants precultured on stepwise concentrations of sucrose and subsequent dehydration in PVS3 for 30 min. Cryopreserved shoot tips were regenerated to whole plants using pre-optimized conditions of micropropagation. This study confirms the potential of TIS for micropropagation in ex situ conservation and reintroduction of endangered American chestnuts and possibly other woody plant species.

Yukon Draba (Draba yukonensis) is a small, short-lived perennial mustard species that is endemic to southwestern Yukon in Canada. This plant has been categorized as a species of Special Concern. It faces the threat of habitat loss due to natural and man-made causes and a population that is unevenly distributed to a few large and several small subpopulations in the area. It will therefore be judicious to undertake investigations on the conservation of this species to save it from further deterioration which may lead to its extinction. In this study, a protocol was developed for in vitro propagation and cryopreservation of Yukon Draba. The micropropagation protocol was optimized using shoot tips which enabled clonal propagation and in vitro storage of the species. Shoots grew best in the medium containing MS basal salts and had the highest multiplication with the addition of 2 µM 6-benzylaminopurine or 5 µM Kinetin with 3% sucrose. The addition of 10 µM Indole Butyric Acid (IBA) produced the highest number of adventitious roots on the shoots and the longest root length was observed at 2 µM IBA. The rooted plantlets were transferred to greenhouse and the highest survival (87.5%) was observed for the plantlets treated with a lower concentration of IBA (2 µM). Cryopreservation protocol was developed using the droplet-vitrification method for in vitro shoot tips. Two-week-old shoots had the highest survival and regrowth following exposure to plant vitrification solution 3 (PVS3) for 30 min, prior to direct immersion of the droplets into the liquid nitrogen. The optimized protocols for the micropropagation and cryopreservation may be useful for the long-term germplasm conservation and reintroduction of this species in its natural habitat.