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Shoot Tips

Cryopreservation of shoot tips is one option for species lacking seeds or for species with recalcitrant seeds for which the zygotic embryos cannot be cryopreserved. Several methods are available including droplet vitrification (Panis et al., 2005), vitrification and encapsulation vitrification (Sakai et al., 2008), encapsulation dehydration (Fabre ad Dereuddre, 1990), and slow-freezing or two step freezing (Volkova et al., 2015), all of which have been successful with some species. There are many examples in the literature of shoot-tip cryopreservation of both exceptional and non-exceptional species that can be helpful when developing a new shoot tip cryopreservation protocol, and a few are selected below. A good starting point is Plant Cryopreservation: A Practical Guide (Reed, 2008). Such protocols are dependent on the in vitro methods that are required for initiating the shoot-propagating culture as well as for recovering plants from cryopreserved shoot tips, and thus, the extensive literature on micropropagation can also be useful.

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Engelmann F (2011) Use of biotechnologies for the conservation of plant biodiversity. In Vitro Cell Dev Biol – Plant 47:5–16. doi: 10.1007/s11627-010-9327-2

Fabre J, Dereuddre J (1990) Encapsulation-dehydration: A new approach to cryopreservation of Solanum shoot tips. CryoLetters 11:413–426.

Folgado R, Panis B. (2019) Cryopreservation of Ashe magnolia shoot-tips by droplet-vitrification. Proc. III International Symposium on Plant Cryopreservation. Acta Hortic 1234: 233-240. https://doi.org/10.17660/ActaHortic.2019.1234.31

Hernández-Ramírez F, Dolce N, Flores-Castaños O, Rascón-Díaz MP, Ángeles-Álvarez G, Folgado R, Gonzalez-Arnao MT. (2020) Advances in cryopreservation of vanilla (Vanilla planifolia Jacks.) shoot-tips: assessment of new biotechnological and cryogenic factors. In Vitro Cell Dev Biol-Plant 56. https://doi.org/10.1007/s11627-020-10069-w

Jenderek MM, Reed BM (2017) Cryopreserved storage of clonal germplasm in the USDA National Plant Germplasm System. In Vitro Cell Dev Biol – Plant 53:1-10

Kaczmarczyk A, Funnekotter B, Turner SR, et al (2013) Development of cryopreservation for Loxocarya cinerea – An endemic Australian plant species important for post-mining restoration. Cryo-Letters 34:508–519.

Normah MN, Sulong N, Reed BM (2019) Cryopreservation of shoot tips of recalcitrant and tropical species: Advances and strategies. Cryobiology 87:1–14. doi: 10.1016/j.cryobiol.2019.01.008

O’Brien C, Hiti-Bandaralage J, Folgado R, Lahmeyer S, Hayward A, Folsom J, Mitter N. (2020) A method to increase regrowth of vitrified shoot tips of avocado (Persea americana Mill.): First critical step in developing a cryopreservation protocol. Sci Hortic (Amsterdam) 266:1-6. https://doi.org/10.1016/j.scienta.2020.109305

O’Brien C, Hiti-Bandaralage J, Folgado R, Hayward A, Lahmeyer S, Folsom J, Mitter N. (2021)  Cryopreservation of woody crops: The avocado case. Plants, 10: 934 https://doi.org/10.3390/plants10050934

O’Brien C, Hiti-Bandaralage JCA, Folgado R, Lahmeyer S, Hayward A, Folsom J, Mitter N. (2021) First report on cryopreservation of mature shoot tips of two avocado (Persea americana Mill.) rootstocks. Plant Cell Tiss Organ Cult, 144: 103–113. https://doi.org/10.1007/s11240-020-01861-y

O’Brien C, Hiti-Bandaralage JCA, Folgado R, Lahmeyer S, Hayward A, Mitter N. (2020) Developing a cryopreservation protocol for avocado (Persea americana Mill.) apical shoot tips using different antioxidants. Acta Hortic. 1285: 15-22.  https://doi.org/10.17660/ActaHortic.2020.1285.3

O’Brien, C., Hiti-Bandaralage, J., Folgado, R., Lahmeyer, S., Hayward, A., Folsom, J. and Mitter, N. (2020). A method to increase regrowth of vitrified shoot tips of avocado (Persea americana Mill.): First critical step in developing a cryopreservation protocol. Scientia Horticulturae, 266, (2) 109305. https://doi.org/10.1016/j.scienta.2020.109305

Panis B, Piette B, Swennen R (2005) Droplet vitrification of apical meristems : a cryopreservation protocol applicable to all Musaceae. Plant Sci 168:45–55. doi: 10.1016/j.plantsci.2004.07.022

Park S-U, Kim H-H (2016) Cryopreservation of sweet potato shoot tips using a droplet- vitrification procedure. CryoLetters 36:344–352.

Pence VC, Chaiken M (2021) Shoot tip cryopreservation as a conservation tool for species of Quercus: Effect of species and environment on recovery. Cryo-Letters 42: 159-167.

Pence VC, Philpott M, Culley TM, et al (2017) Survival and genetic stability of shoot tips of Hedeoma todsenii R.S.Irving after long-term cryostorage. In Vitro Cell Dev Biol – Plant 53:329–338. doi: 10.1007/s11627-017-9854-1

Pokorna E, Faltus M, Machova P, et al (2020) Grey poplar explant acclimation to improve the dehydration tolerance and cryopreservation. Biol Plant 64:119–128. doi: 10.32615/bp.2019.148

Rathwell R, Popova E, Shukla MR, Saxena PK (2016) Development of cryopreservation methods for cherry birch (Betula lenta L.), an endangered tree species in Canada. Can J For Res 46:1284–1292. doi: 10.1139/cjfr-2016-0166

Reed BM (ed) (2008) Plant Cryopreservation: A Practical Guide. Springer, New York

Sakai A, Hirai D, Niino T (2008) Development of PVS-based vitrification and encapsulation-vitrification protocols. In: Reed BM (ed) Plant Cryopreservation: A Practical Guide. Springer, pp 33–57

San José MC, Valladares S, Vázquez-Janeiro L, Corredoira E  (2014). Cryopreservation of in vitro-grown shoot-tips of Alnus glutinosa (L.) Gaertn. by vitrification. Acta Physiologia Plantarum 36:109-116

San José MC, Janeiro LV, Corredoira E (2015) Simple strategy for the in vitro conservation of Alnus glutinosa (L.) Gaertn. germplasm. Trees-Structure and Function 29: 539-549.

Sharma N, Gowthami R, Vimala S, et al (2020) Cryopreservation of shoot tips of Gentiana kurroo Royle–a critically endangered medicinal plant of India. Plant Cell Tissue Organ Cult. doi: 10.1007/s11240-020-01879-2

Touchell DH, Dixon KW, Tan B (1992) Cryopreservation of shoot-tips of Grevillea scapigera (Proteaceae): a rare and endangered plant from Western Australia. Aust J Bot 40:305–310.

Turner SR, Senaratna T, Bunn E, et al (2001) Cryopreservation of shoot tips from six endangered Australian species using a modified vitrification protocol. Ann Bot 87:371–378. doi: 10.1006/anbo.2000.1346

Uchendu EE, Reed BM (2008) A comparative study of three cryopreservation protocols for effective storage of in vitro-grown mint {Mentha spp.). Cryo-Letters 29:181–188.

Vidal N, Sánchez C, Jorquera L, et al (2005) Cryopreservation of chestnut by vitrification of in vitro-grown shoot tips. In Vitro Cell Dev Biol – Plant 41:63–68. doi: 10.1079/IVP2004596

Volk GM, Walters C (2006) Plant vitrification solution 2 lowers water content and alters freezing behavior in shoot tips during cryoprotection. Cryobiology 52:48–61. doi: 10.1016/j.cryobiol.2005.09.004

Volkova LA, Urmantseva V V., Popova E V., Nosov AM (2015) Physiological, cytological and biochemical stability of Medicago sativa L. cell culture after 27 years of cryogenic storage. Cryo-Letters 36:252–263.

Vollmer R, Villagaray R, Egúsquiza V, et al (2016) The Potato Cryobank at the International Potato Center ( CIP ): A model for long term conservation of clonal plant genetic resources collections of the future. CryoLetters 37:318–329.

Wilkinson T, Wetten A, Prychid C, Fay MF (2003) Suitability of cryopreservation for the long-term storage of rare and endangered plant species: A case history for Cosmos atrosanguineus. Ann Bot 91:65–74. doi: 10.1093/aob/mcg009

Zhang XC, Bao WW, Zhang A-ling, et al (2020) Cryopreservation of shoot tips, evaluations of vegetative growth, and assessments of genetic and epigenetic changes in cryo-derived plants of Actinidia spp. Cryobiology. doi: 10.1016/j.cryobiol.2020.05.004

Zhao L, Wang M, Li J, et al (2019) Cryobiotechnology: A double-edged sword for obligate plant pathogens. Plant Dis 103:1058–1067. doi: 10.1094/PDIS-11-18-1989-FE

Video Resources

The potential of shoot tip cryopreservation as a conservation tool for oaks (Quercus spp.) by Valerie Pence

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Valerie C. Pence is the Director of Plant Research at the Center for Conservation and Research of Endangered Wildlife (CREW) at the Cincinnati Zoo & Botanical Garden. Her work focuses on applying in vitro methods and cryobiotechnologies to the conservation of exceptional species. Her research has included developing protocols for the cryo-conservation of shoot tips, embryos, gametophytes, spores, seeds, and pollen of species from across the U.S., many critically endangered.

Cryopreservation of rare and threatened Australian species shoot tips by Bryn Funnekotter


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Bryn Funnekotter is a Research Scientist at Curtin University and Kings Park Science, Biodiversity Conservation Science, DBCA, Western Australia. Bryn’s research focuses on cryopreserving rare and threatened Australian native plants, with a focus on understanding the stresses imposed by the cryopreservation process.

Cryobiotechnology tools to conserve shoot tips from exceptional species by Raquel Folgado


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Dr. Raquel Folgado serves as a cryopreservation botanist at The Huntington Botanical Gardens (Southern California, USA). She is leading a cryobiotechnology program that involves research projects on avocado and wild plants, including aloes, agaves, cacti, magnolias, cycads, and oaks. Dr. Folgado has created in vitro repositories at The Huntington to preserve and allow the distribution of Persea, Aloe, Agave, Magnolia species, and some hybrids with a commercial interest. One of her goals is to design protocols to preserve plant germplasm in liquid nitrogen to ensure the long-term conservation of threatened and rare species. Dr. Folgado’s interest extends to a better knowledge of the tolerance to abiotic constraints, which will help standardize cryopreservation methods. Methods have been optimized for many crops, but attention is needed for exceptional plant species and specialty crops like avocado. Dr. Folgado is associated with scientific societies and serves as a reviewer for several scientific journals; she collaborates with researchers worldwide to exchanging knowledge and skills in cryobiotechnology and plant abiotic stress.

Improving survival rates after cryopreservation due to genetic variation and tissue age in exceptional Hawaiian species Melicope mucronulata by Maribeth Stafford, Megan Philpott, and Valerie Pence

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Maribeth Stafford was a plant conservation research intern at the Cincinnati Zoo & Botanical Garden’s Center for Conservation Research and Endangered Wildlife (CREW) for three years. She worked with Dr. Megan Philpott to bank CREW’s collection of exceptional Hawaiian species in collaboration with the Lyon Arboretum in Honolulu. Maribeth was awarded a grant to aid her research on Melicope mucronulata by Botanic Gardens Conservation International (BGCI) in 2021. She completed her Bachelor of Science degree in Biology at the University of Cincinnati in 2021 and is currently studying to become a medical doctor.

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