210 W. A. VENDRAME AND A. A. KHODDAMZADEH
Early reports on pollen storage indicated that fresh pollen from differ-
ent orchid species and hybrids could be preserved in ultra-low tempera-
ture (–79◦C), but moderate desiccation was required for proper recovery
(Ito 1965). More recently, Vendrame et al. (2008) reported the cryop-
reservation of pollen from twoDendrobiumunregistered hybrids; “Sena
Red Thailand” and “Mini W/RL.” The effectiveness of cryopreservation
was assessed by pollinating flowers of the same hybrids with pollen
that had been submitted to the different cryopreservation treatments.
However, no significant differences were found among the different
treatments and the controls for the two orchid hybrids. Pollination of
flowers with cryopreserved pollen from all treatments and controls was
successful with the subsequent formation of capsules and viable seeds.
Therefore, for the selected orchid hybrids, cryopreservation of pollen
can be achieved by its simple and direct storage in liquid nitrogen.
E. Meristems and Shoot Apices
The pre-treatment of plant tissues and organs with cryoprotective sub-
stances, such as dimethyl sulfoxide (DMSO), ethylene glycol, methanol,
glycerol, and propylene glycol, among others is important for the
proper survival and recovery of cryopreserved plant material. In some
instances and depending on tissues used, a gradual and slow freezing
is performed. Temperature is dropped gradually at a rate between 1◦C
and 10◦C per hour until it reaches about –40◦C, prior to immersion in
liquid nitrogen. Small tissues that exhibit uniform morphology, such
as suspended cell cultures and calli are easier to cryopreserve as com-
pared larger tissues, such as zygotic and somatic embryos, and shoots.
Although such tissues could be of interest for orchid cryopreservation,
studies are quite limited. Some of the few studies available to date
include the cryopreservation of shoot primordia inVanda pumila(Hai-
Yan and Kondo 1996) and calli obtained from apical meristems ofPha-
laenopsis, as reported by (Tsukazaki et al. 2000).
More recently, different tissues and organs from several orchids have
been successfully cryopreserved, as reported by Gonzalez-Arnao et al.
(2014) and cryotherapy has been suggested as a means for eliminating
plant pathogens, a technique that could assist in the long-term storage
associated with production of clean orchid plant material.
VI. SUMMARY AND CONCLUSIONS
Biotechnology continues to offer valuable tools for orchid produc-
tion and conservation. Advances in orchid propagation have allowed