Cryopreservation of temperate recalcitrant seed

Study of the fundamental basis of desiccation and low temperature stress tolerance in recalcitrant seed tissues of temperate species in order to optimise their cryopreservation.

Sweet chestnut

Several important European tree genera produce recalcitrant seeds that, due to their desiccation sensitivity, cannot be stored in conventional seed bank conditions of low humidity and temperature. These include the oaks (Quercus) and chestnuts (Castanea, Aesculus). As part of the Global Tree Seed Bank Project, we will study recalcitrant seed tolerance to the stresses of partial desiccation, cryoprotectants and low temperatures. The information gathered will help to develop generic protocols for banking recalcitrant seeds, and will help us to conserve these species, many of which are of significant conservation interest.

Seed recalcitrance, a challenge for ex situ conservation in seed banks

Long-term preservation of seeds at the standard conditions of seed banks (dried to low moisture contents and stored at low temperatures) is only feasible for orthodox (desiccation-tolerant) seeds. However, a large number of species cannot be preserved under these conditions, as is the case for species producing desiccation sensitive (recalcitrant) seeds. About 8% of the seeded plants of the world produce recalcitrant seeds. Since the proportion of recalcitrant seeds is habitat dependent, recalcitrance becomes more relevant in particular habitats, such as tropical evergreen rain forests. Here it has been suggested that ca. 19% of the indigenous flora may produce recalcitrant seeds, a proportion that increases to c. 47% when looking at trees and shrubs.

Cryobiotechnology, the challenging solution

Cryopreservation (the process of using sub-zero temperatures to preserve living cells and tissues) offers a potential long-term storage solution for recalcitrant seeds, however, only a small number of species have well established and successful cryopreservation protocols. This is partly because desiccation sensitivity and responses to cryopreservation procedures are variable, and need to be ascertained per species as well as genotype.

Research lead solutions

In this research, desiccation sensitivity of various recalcitrant seeds and their response to innovative cryopreservation protocols, such as “vacuum infiltration vitrification (VIV)”, is being investigated. A diversity of oak species (Quercus sp.), as well as other common species of European forests producing recalcitrant seeds – e.g. sweet chestnut (Castanea sativa) or horse-chestnut (Aesculus hippocastanum) – will benefit from this research. The main aim of this research is to develop a generic protocol for the banking of recalcitrant seeds, mainly focussing on the cryopreservation of the embryonic axis. The responses of diverse species will be investigated in this comparative study, in the context of known mechanisms of tree seed desiccation (in)tolerance.

Why Kew?

Kew hosts the largest seed bank for wild species in the world, the Millennium Seed Bank. Currently, the Seed Bank is focused on the conservation of orthodox seeds. However, research into the cryopreservation of recalcitrant seeds could provide the potential for conserving many more species.


  • To characterise desiccation sensitivity of the selected species at the seed and embryo level across an environmental cline.
  • To define the optimum explant for cryopreservation and route (pre- and post-cooling treatments) to maximum recovery in vitro for the selected species.
  • To develop a generic cryopreservation protocol for temperate recalcitrant species, which will serve as a roadmap to tackling the relatively more intractable tropical species.
  • To study desiccation sensitivity in tissues of a range of recalcitrant seeded species.
  • Identify of the most appropriate plant tissue to use in cryopreservation and recovery in vitro (sterile environment).
  • Search for a generic cryopreservation solution to the storage of recalcitrant seeded tissues.
  • Disseminate results to scientific and general audience.


  • Ministerio de agricultura y pesca, alimentación y medio ambiente, Spain.


  • Forest Research

Garfield Weston Foundation

Pritchard, H.W., Nadarajan, J., Ballesteros, D., Thammasiri, K., Prasongsom, S., Malik, S.K., Chaudhury, R., Kim, H-H., Lin, L., Li, W-Q., Yang, X-Y. & Popova E. (2017)

Cryobiotechnology of tropical seeds – scale, scope and hope

Acta Horticulturae 1167: 37-47

Pence, V.C., Philpott, M., Culley, T.M., Plair, B., Yorke, S.R., Lindsey, K., Vanhove, A-C. & Ballesteros, D. (2017)

Survival and genetic stability of shoot tips of Hedeoma todsenii after long-term cryostorage

In Vitro Cellular & Developmental Biology – Plant 53:328–338. DOI: 10.1007/s11627-017-9854-1

Ballesteros, D. & Pence, V.C. (2017)

Survival and death of seeds during liquid nitrogen storage: a case study on seeds with short lifespans

Cryoletters 38(4), 278-289

Merrett-Wade, E., Nadarajan, J., Yang, X., Ballesteros, D., Sun, W., & Pritchard, H.W. (2016)

Plant species with extremely small populations (PSESP) in China: a seed and spore biology perspective

Plant Diversity, 38(5): 209–220

Nadarajan, J. & Pritchard, H.W. (2014)

Biophysical characteristics of successful oilseed embryo cryoprotection and cryopreservation using vacuum infiltration vitrification, an innovation in plant cell preservation

PLoS One. DOI: 10.1371/journal.pone.0096169 e0096169