oven for 16–17 h at 103
C. The dry weight is measured on the
microbalance after cooling the embryos for 10 min to room
temperature in a dry box containing activated silica gel.
26. The drying method showed in this protocol works well for
diverse species that have been researched at Kew and other
species found in the literature (e.g., Aesculus hippocastanum,
A. californica, Aesculus  arnoldiana, Quercus pyrenaica,
Q. rubra). However, for some other species, the use of cryoprotectants such as glycerol and sucrose before the drying step
is needed (e.g., Amaryllis belladonna [44]). Another cryopreservation method using vacuum infiltration of the cryoprotectant Plant Vitrification Solution 2 (PVS2) has been developed
for embryonic axes of oily seeds, including the recalcitrant
seeds of Laurus nobilis [45]. While the development of protocols using cryoprotectants may be necessary for diverse species,
partial drying and fast cooling as exposed in this chapter is
recommended as the first method to try in a recalcitrant-seeded
species with unknown response to cryopreservation.
Acknowledgments
We thank diverse researchers and technicians from RBG Kew and
the MSB that have supported and revised the development of these
protocols, particularly John Adams for helping to build the “flash
drier” used at RBG Kew and Frances Stanley and Kirstine Manger
for making a critical review of the manuscript. RBG Kew receives
grant-in-aid from Defra.
References
1. Li DZ, Pritchard HW (2009) The science and
economics of ex situ plant conservation.
Trends Plant Sci 14:614–621
2. Roberts EH (1973) Predicting the storage life
of seeds. Seed Sci Technol 1:499–514
3. Walters C (2015) Orthodoxy, recalcitrance and
in-between: describing variation in seed storage characteristics using threshold responses to
water loss. Planta 242:397–406
4. Walters C, Berjak P, Pammenter N, Kennedy K,
Raven P (2013) Preservation of recalcitrant
seeds. Science 339:915–916
5. Wesley-Smith J, Berjak P, Pammenter NW,
Walters C (2014) Intracellular ice and cell survival in cryo-exposed embryonic axes of recalcitrant seeds of Acer saccharinum: an
ultrastructural study of factors affecting cell
and ice structures. Ann Bot 113:695–709
6. FAO (2014) Genebank standards for plant
genetic resources for food and agriculture,
Rev. FAO, Rome
7. Center for Plant Conservation (2019) CPC
Best plant conservation practices to support
species survival in the wild. Center for Plant
Conservation, Escondido, CA
8. Millennium Seed Bank (MSB) Partnership
(2015) Seed conservation standards for MSB
partnership collections. Royal Botanic Gardens, Kew, UK
9. Wyse SV, Dickie JB (2017) Predicting the
global incidence of seed desiccation sensitivity.
J Ecol 105:1082–1093
680
Daniel Ballesteros et al.
C. The dry weight is measured on the
microbalance after cooling the embryos for 10 min to room
temperature in a dry box containing activated silica gel.
26. The drying method showed in this protocol works well for
diverse species that have been researched at Kew and other
species found in the literature (e.g., Aesculus hippocastanum,
A. californica, Aesculus  arnoldiana, Quercus pyrenaica,
Q. rubra). However, for some other species, the use of cryoprotectants such as glycerol and sucrose before the drying step
is needed (e.g., Amaryllis belladonna [44]). Another cryopreservation method using vacuum infiltration of the cryoprotectant Plant Vitrification Solution 2 (PVS2) has been developed
for embryonic axes of oily seeds, including the recalcitrant
seeds of Laurus nobilis [45]. While the development of protocols using cryoprotectants may be necessary for diverse species,
partial drying and fast cooling as exposed in this chapter is
recommended as the first method to try in a recalcitrant-seeded
species with unknown response to cryopreservation.
Acknowledgments
We thank diverse researchers and technicians from RBG Kew and
the MSB that have supported and revised the development of these
protocols, particularly John Adams for helping to build the “flash
drier” used at RBG Kew and Frances Stanley and Kirstine Manger
for making a critical review of the manuscript. RBG Kew receives
grant-in-aid from Defra.
References
1. Li DZ, Pritchard HW (2009) The science and
economics of ex situ plant conservation.
Trends Plant Sci 14:614–621
2. Roberts EH (1973) Predicting the storage life
of seeds. Seed Sci Technol 1:499–514
3. Walters C (2015) Orthodoxy, recalcitrance and
in-between: describing variation in seed storage characteristics using threshold responses to
water loss. Planta 242:397–406
4. Walters C, Berjak P, Pammenter N, Kennedy K,
Raven P (2013) Preservation of recalcitrant
seeds. Science 339:915–916
5. Wesley-Smith J, Berjak P, Pammenter NW,
Walters C (2014) Intracellular ice and cell survival in cryo-exposed embryonic axes of recalcitrant seeds of Acer saccharinum: an
ultrastructural study of factors affecting cell
and ice structures. Ann Bot 113:695–709
6. FAO (2014) Genebank standards for plant
genetic resources for food and agriculture,
Rev. FAO, Rome
7. Center for Plant Conservation (2019) CPC
Best plant conservation practices to support
species survival in the wild. Center for Plant
Conservation, Escondido, CA
8. Millennium Seed Bank (MSB) Partnership
(2015) Seed conservation standards for MSB
partnership collections. Royal Botanic Gardens, Kew, UK
9. Wyse SV, Dickie JB (2017) Predicting the
global incidence of seed desiccation sensitivity.
J Ecol 105:1082–1093
680
Daniel Ballesteros et al.