- ORCHID BIOTECHNOLOGY 201
Reed 2008). These includeCentaurium rigualii(Gonzalez-Benito and
Perez 1997), alfalfa (Shibli et al. 2001), hop and pelargonium (Dumet
et al. 2002), citrus (Gonzalez-Arnao et al. 2003), grapevine (Wang et al.
2004), cocoa (Fang et al. 2008),Melia azedarach(Scocchi et al. 2004),
gentian (Suzuki et al. 2005), and raspberry (Wang et al. 2005), among
others.
A wide range of orchids have already been adapted to cryopreser-
vation, such asBletilla striata (Ishikawa et al. 1997; Hirano et al.
2005; Jitsopakul et al. 2008),Vanda pumila(Na and Kondo 1996),Pha-
laenopsis(Tsukazaki et al. 2000), andDendrobium species and hybrids
(Lurswijidjarus and Thammasiri 2004; Carvalho 2006; Vendrame et al.
2007b; Galdiano et al. 2012, 2014), andOncidium flexuosum(Galdiano
et al. 2013), among many others. Several cryopreservation methods for
orchid seeds, pollen, protocorms, meristems, and shoot apices have
been reviewed and are reported for many different species and hybrids
(Vendrame et al. 2014). Table 4.2 lists the most relevant cryopreser-
vation studies with orchids. Next we discuss some cryopreservation
techniques used for orchid seeds, protocorms, pollen, and meristematic
issues.
B. Seeds
Orchid seed storage is an important aspect for long-term conservation
of seeds from different orchid species, allowing preservation of endan-
gered material with little space required, facilitating germplasm storage
and distribution at low cost (Pritchard and Seaton 1993), and allow-
ing storage of material for breeding and genetic improvement programs
(Vendrame et al. 2007b). In general, seeds from various orchid species
can be maintained under conventional storage methods as those used
for other seeds (–18◦C and 5% humidity). However, seed viability and
longevity are highly variable and traditional methods are not recom-
mended for long-term seed conservation (Pritchard and Seaton 1993).
Various studies have been conducted in recent years to determine the
best storage conditions for orchid seeds. The results are highly variable
depending on the method used, the species, and even the lot of seeds
tested (Vendrame et al. 2014).
Early studies with seeds from 30 different orchid species stored
at –10◦C showed that viability remained for 3 years, but declined
over 10 years of storage, when all seeds were dead (Bowling and
Thompson 1972; Pritchard 1985). However, a study on storage of seeds
from several orchid species and hybrids in ultra-low temperatures
(–79◦C) indicated that seeds should be fresh, but moderate desiccation
prior to storage was essential for successful recovery (Ito 1965). Early