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Bratoniahybrid seeds. Best results were obtained in MS and Morel
media (Morel 1960).
The use of cryoprotectants prior to immersion in liquid nitrogen is
also essential for the proper cryopreservation of protocorms. The cryop-
reservation of protocorms ofD. nobilewas evaluated using a combina-
tion of cryoprotectants in seven treatments (Vendrame and Faria 2011).
The best recovery, survival (68%), and development of protocorms in
this orchid species was obtained when using a 2 M glycerol solution for
20 min added to a vitrification (PVS2) solution with 1% phloroglucinol
for 10 min. Phloroglucinol combined with glycerol and PVS2 showed
a positive effect. However, the addition of 0.04 M sucrose to the treat-
ments showed a significant reduction in survival after cryopreservation
(Vendrame and Faria 2011).
Galdiano et al. 2012 also evaluated the cryopreservation ofDen-
drobiumhybridD. Swartz“Dong Yai” protocorms using two cryopro-
tectants, phloroglucinol and Supercool X1000. Protocorms were pre-
treated with 0.3 M sucrose for 24 h, followed by exposure to PVS2 solu-
tion containing either 1% phloroglucinol or 1% Supercool X1000 for
15 min at 0◦C prior to cryopreservation. After 75 d, protocorm survival
was higher and similar to the initial germination rates for treatments
using 1% phloroglucinol alone.
D. Pollen
Long-term pollen storage is of importance for the conservation of
genetic diversity and for use in plant breeding programs. Pollen of
rare, threatened, and/or endangered species can also be preserved in
germplasm banks created with pollen (Connor and Towill 1993; Grout
and Roberts 1995; Sacks and St. Clair 1996; Reed 2008). Furthermore,
pollen storage allows the preservation of large amounts of genetic mate-
rial (Towill 2002). Although Pritchard et al. (1999) recommend cryop-
reservation of seeds and pollen for the creation of orchid germplasm
banks, they suggest further research is needed.
Orchid production and reproduction depends on proper pollen stor-
age, which enables crosses between plants that exhibit temporal and
spatial separation in their periods of sexual reproduction (Vendrame
et al. 2008). In addition, pollen storage can assist in the development
of haploid plants through the development of embryos from pollen
grains (Pritchard and Prendergast 1989). Temperatures for pollen stor-
age varies from –20, –4 to+ 4 ◦C, and as intracellular water levels are
maintained low, lower storage temperatures provide greater longevity
for pollen and also seeds (Towill 2002).