Conservation of cactus pollen by freezing History 1955–Tijs Visser (Wageningen, NL) shows that frozen pollen of pear and apple trees germinates better when its water content was reduced prior to freezing at –180°C. 1990 – Charrier demosntrates with frozen pollen in cereals that the integrity of the glycoproteins of the pollen’s outer envelope is indispensable for the recognition by the stigma. 1995 – Consensus that water content in pollen should be at 2 % to 5 % before freezing. Conservation of cactus pollen by freezing History(cont.) 1998-2000 – Julia Buitink demonstrated that vitrification (freezing without formation of ice crystals) of its innercellular content is necessary for maintaining the pollen’s viability. 2000 – Metz, Nerd & Mizrahi publish a technique for the dehydration of pollen from Hylocereus undatus and H. polyrhizus which have different main blooming seasons. Pollen harvested in the afternoon contains more water than pollen from the morning (48 % vs 22 %). Conservation of cactus pollen by freezing Dehydration under vacuum according to Metz 2000 Hylocereus The technique % Exposing the pollen to a partial vacuum is necessary in order to Afternoon pollen pollen, of reduce the water content below 5 % before freezing. Morning pollen content The pollen is rehydrated Water naturally on contact with the stigma after pollination. Time, hours Conservation of cactus pollen by freezing Building a desiccator (a container for drying by vacuum) Essential: manometer for control of the vacuum Two bicycle v a l v e s A glas for jam or similar that can be sealed hermetically Conservation of cactus pollen by freezing Ways o f c reating a vaccum i n t h e desiccator A vacuum pump for wine bottles A syringe A compressor from a refrigerator Jean-Didier Hary (Translator’s comment: It’s not recommended to disassemble an old refrigerator on your own. The cooling fluid is to be discarded by an expert.) Conservation of cactus pollen by freezing Collecting pollen A funnel for large flowers Scissors for harvesting the anthers Pollen extracted by suction through a half cotton bud Conservation of cactus pollen by freezing Dehydrating the pollen Pollen in a glassine paper bag Air pressure in desiccator reduced by 50 % (to ½ atmosphere) Vacuum maintained for 4 hours at room temperature Silica gel for adsorption of remaining humidity Conservation of cactus pollen by freezing Freezing the pollen Pollen is frozen in a glassine paper bag in a hermetically sealing container (e.g. a glas for jam or similar) together with silica gel. It can be kept frozen this way for several years without loosing its viability. Conservation of cactus pollen by freezing Bibliography Buitink J., W a l t e r s C., Hoekstra F.A. & Crane J., 1998a, Storage behavior of Typha latifolia pollen at low w a t e r contents : interpretation on the basis of w a t e r activity and glass concepts., Physiologia Plantarum, 103 : 145– 153. Buitink J., Claessens M.A.E., Hemminga M.A., Hoekstra F.A., 1998b, Influence of w a t e r content and temperature on molecular mobility and intracellular glasses in seed and pollen, Plant Physiol., 118 : 531-541 BuitinkJ., Leprince O., Hemminga M. A., Hoekstra F. A., 2000, The effects of moisture and temperature on the ageing kinetics of pollen : interpretation based on cytoplasmic mobility, Plant, Cell & Environment, 23, 9 : 967–974 Cerceau-Larrival M.-Th. , Delange Y. , Y o u m b i E. , Derouet L. , Verhille A.-M. and Carbonnier-Jarreau M.-C., 1995, Contribution à la préservation du patrimoine génétique mâle des collections végétales vivantes du Muséum National d'Histoire Naturelle, P a r i s , Grana, 34, 6 : 371 — 407 Conservation of cactus pollen by freezing Bibliography (cont.) Charrier A., 1990, P o l l e n et ressources génétiques, Bull. Soc. Bot. F r . , 2 : 101-104. Derouet M., 2010, Graines de cactées, de la fleur à la plantule, page 18. http://www.cactuspro.com/articles/graines_de_cactees_de_la_fleur_a_la_plantule Metz C., Nerd A., Mizrahi Y., 2000, Viability of pollen of two fruit crop cacti of the genus Hylocereus is affected b y temperature and duration of storage, HortScience, 35, 2 : 199-201 Visser T., 1955, Germination and storage of pollen, Thesis, Mededelingen v a n de Landbouwhogeschool 55 (1) : 68p, NL Conservation of cactus pollen by freezing Summary Long-term storage of pollen is interesting f o r cacti enthusiasts seeking crosses. This paper popularize a home-made experiment based on the study developped b y Metz et al. (2000). T o obtain an efficient preservation of viable pollen, two processes are required : first dehydration in a vacuum (about -50kPa) dessicator at room temperature until the moisture content is r e d u c e d t o 5% (about 4 hours) and second, storage at subfreezing temperature. In practice, the vacuum is performed b y s y r i n g e , vacuum wine saver pump or refrigerating unit and the storage in a household freezer. F o r pollination, the pollen is applied t o mature stigmas able t o r e h y d r a t e the dried pollen. Under these conditions, w e obtain fruits and seeds after many y e a r s of storage. Conservation of cactus pollen by freezing Conclusion This technique is easy to apply and permits creation of hybrids at any time. Acknowledgements Georges Marchand (†) and Jean-Didier Hary for their kind collaboration. The original article’s PDF file (updated 2014) can be downloaded from the Cactus Francophone website: https://www.cactuspro.com/articles/conservation-du-pollen-par-congelation [email protected] 9043 Cleistocactus smaragdiflorus X Lobivia pugionacantha.