4 Notes
1. The use of saturated salt solutions (e.g., MgCl 2 ) or dissolutions
of LiCl [37] can be an alternative to generate an environment
of 30% RH in a sealed jar when controlled environment chambers are not available.
2. Due to the short life span of chlorophyllous spores, avoid
drying them for too long. During the first day of drying,
water content of the fern spores will be reduced significantly
[38]. In 2 or 3 days of drying at 30% RH, water content may
not be in equilibrium with the ambient RH but will decrease
enough to “safe levels” for cryopreservation [36]. It is a matter
of balance between water content adjustment to low RHs and
viability lost due to aging in the dry state [8].
3. A pollen “flash drier” is a device used to dry pollen very fast in a
stream of dry air at room temperature (about 20
C). It can be
made using different designs (see Fig. 2). Barnabas [41] used a
vertical glass cylinder of 30 cm with a filter on each side that was
placed in a dry cabinet or chamber (set at a RH of 20–40%). In
this design (see Fig. 2a), the air must be blown into the tube
from below, so the pollen stays in flotation and dries uniformly.
Buitink et al. [42] used a device similar to that described for
embryonic axes in Chapter 36. Basically, consists in a computer
fan that blows the air into two layers of a mesh containing the
pollen grains (e.g., see Fig. 2b). The fan is inside a jar that
contains activated silica gel in the bottom. The pore size of
the filter or the mesh must not exceed the pollen diameter
considering that this can be reduced up to 50% during drying.
4. Fern spores are produced in structures called sporangia which
are generally grouped in structures called sorus located underneath the frond (except for some species that produce the
spores in sporangia enclosed in transformed leaves known as
fertile leaves or strobili in the case of Equisetum sp.). Sorus and
sporangia are easily visible with the naked eye, and sporangia
are often confused with the spores, but their detailed structure
is revealed using a simple Â10 hand lens or microscope. Some
sorus are circular and others linear, may or may not be protected by a membranous outgrowth on the undersurface of the
fern leave called indusium, and come in a variety of colors. The
color of the sorus or the indusium can be indicative of the
maturity stage of the spore (see ref. 5 for detailed information).
However, most sorus tend to be pale green when immature (see
Fig. 1a), progressing to a brown/blackish color when ripe (see
Fig. 1b), except for the fertile fronds of most chlorophyllous
spores that change from pale or light green when immature to
Fern Spores and Pollen Cryopreservation
631
1. The use of saturated salt solutions (e.g., MgCl 2 ) or dissolutions
of LiCl [37] can be an alternative to generate an environment
of 30% RH in a sealed jar when controlled environment chambers are not available.
2. Due to the short life span of chlorophyllous spores, avoid
drying them for too long. During the first day of drying,
water content of the fern spores will be reduced significantly
[38]. In 2 or 3 days of drying at 30% RH, water content may
not be in equilibrium with the ambient RH but will decrease
enough to “safe levels” for cryopreservation [36]. It is a matter
of balance between water content adjustment to low RHs and
viability lost due to aging in the dry state [8].
3. A pollen “flash drier” is a device used to dry pollen very fast in a
stream of dry air at room temperature (about 20
C). It can be
made using different designs (see Fig. 2). Barnabas [41] used a
vertical glass cylinder of 30 cm with a filter on each side that was
placed in a dry cabinet or chamber (set at a RH of 20–40%). In
this design (see Fig. 2a), the air must be blown into the tube
from below, so the pollen stays in flotation and dries uniformly.
Buitink et al. [42] used a device similar to that described for
embryonic axes in Chapter 36. Basically, consists in a computer
fan that blows the air into two layers of a mesh containing the
pollen grains (e.g., see Fig. 2b). The fan is inside a jar that
contains activated silica gel in the bottom. The pore size of
the filter or the mesh must not exceed the pollen diameter
considering that this can be reduced up to 50% during drying.
4. Fern spores are produced in structures called sporangia which
are generally grouped in structures called sorus located underneath the frond (except for some species that produce the
spores in sporangia enclosed in transformed leaves known as
fertile leaves or strobili in the case of Equisetum sp.). Sorus and
sporangia are easily visible with the naked eye, and sporangia
are often confused with the spores, but their detailed structure
is revealed using a simple Â10 hand lens or microscope. Some
sorus are circular and others linear, may or may not be protected by a membranous outgrowth on the undersurface of the
fern leave called indusium, and come in a variety of colors. The
color of the sorus or the indusium can be indicative of the
maturity stage of the spore (see ref. 5 for detailed information).
However, most sorus tend to be pale green when immature (see
Fig. 1a), progressing to a brown/blackish color when ripe (see
Fig. 1b), except for the fertile fronds of most chlorophyllous
spores that change from pale or light green when immature to
Fern Spores and Pollen Cryopreservation
631