dehiscence is imminent (so the pollen is shed in the laboratory
at room temperature and RH). For entomophilous species,
single anthers may have to be harvested with forceps to assure
pollen is collected at the optimal maturity. Then anthers will be
dried in the laboratory at room temperature and RH and sieved
to collect the pollen as pollen from entomophilous species tend
to stick to the anther. When pollen is difficult to remove from
the anthers and there are small sample sizes, the whole anther
can be dried and cryopreserved.
15. The viability of the pollen can be assessed by in vitro germination (see Fig. 1i). Each species requires a different medium,
temperature, and photoperiod to germinate. Guidance can be
found elsewhere in the literature [20, 40].
16. Pollen (like some chlorophyllous fern spores; see Note 2) present very short life span, and drying for a long time may reduce
significantly the viability and quality of the pollen cryopreserved. Drying for 24–72 h ensures that pollen is dried to
“safe” moisture contents for cryopreservation and reduces
the change of pollen deterioration.
17. A small sample size is when the amount of pollen/spores
collected fills less than a half of a 2 mL cryovial. A large sample
size is when the amount of pollen collected fills more than a
half of a 2 mL cryovial. Typically, small pollen sample sizes are
collected from entomophilous species, and large sample sizes
can be collected from anemophilous species, particularly trees.
18. Pollen collected in the morning have higher water contents
than pollen collected at noon. This difference can affect to the
drying time and the viability after cryopreservation [41].
19. Appropriate moisture contents for maize cryopreservation
have been empirically determined for diverse cultivars
[41, 43]. The water contents for a higher cryopreservation
success are generally between 0.19 and 0.33 g H 2 O/g dry
weight [14% and 25% moisture content (fresh weight basis)].
At such water contents, maize pollen can retain some viability
and fertility, and water freezing is not detected by DSC
[42]. Moisture contents above 0.40 g H 2 O/g dry weight
[30% moisture content (fresh weight basis)], at which water
freezing transitions are observed by DSC [42], result in lack of
viability after cryopreservation [41]. In some cases, successful
cryopreservation has been detected after drying pollen of some
maize cultivars to water contents between 0.03 and 0.06 g
H 2 O/g dry weight [2.5% and 6.1% moisture content (fresh
weight basis)] [41]. However, maize pollen generally shows a
big decrease in viability when dried <0.40 g H 2 O/g dry
weight and poor or lack of fertility when dried <0.10 g
H 2 O/g dry weight [42].
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Anna Nebot et al.
at room temperature and RH). For entomophilous species,
single anthers may have to be harvested with forceps to assure
pollen is collected at the optimal maturity. Then anthers will be
dried in the laboratory at room temperature and RH and sieved
to collect the pollen as pollen from entomophilous species tend
to stick to the anther. When pollen is difficult to remove from
the anthers and there are small sample sizes, the whole anther
can be dried and cryopreserved.
15. The viability of the pollen can be assessed by in vitro germination (see Fig. 1i). Each species requires a different medium,
temperature, and photoperiod to germinate. Guidance can be
found elsewhere in the literature [20, 40].
16. Pollen (like some chlorophyllous fern spores; see Note 2) present very short life span, and drying for a long time may reduce
significantly the viability and quality of the pollen cryopreserved. Drying for 24–72 h ensures that pollen is dried to
“safe” moisture contents for cryopreservation and reduces
the change of pollen deterioration.
17. A small sample size is when the amount of pollen/spores
collected fills less than a half of a 2 mL cryovial. A large sample
size is when the amount of pollen collected fills more than a
half of a 2 mL cryovial. Typically, small pollen sample sizes are
collected from entomophilous species, and large sample sizes
can be collected from anemophilous species, particularly trees.
18. Pollen collected in the morning have higher water contents
than pollen collected at noon. This difference can affect to the
drying time and the viability after cryopreservation [41].
19. Appropriate moisture contents for maize cryopreservation
have been empirically determined for diverse cultivars
[41, 43]. The water contents for a higher cryopreservation
success are generally between 0.19 and 0.33 g H 2 O/g dry
weight [14% and 25% moisture content (fresh weight basis)].
At such water contents, maize pollen can retain some viability
and fertility, and water freezing is not detected by DSC
[42]. Moisture contents above 0.40 g H 2 O/g dry weight
[30% moisture content (fresh weight basis)], at which water
freezing transitions are observed by DSC [42], result in lack of
viability after cryopreservation [41]. In some cases, successful
cryopreservation has been detected after drying pollen of some
maize cultivars to water contents between 0.03 and 0.06 g
H 2 O/g dry weight [2.5% and 6.1% moisture content (fresh
weight basis)] [41]. However, maize pollen generally shows a
big decrease in viability when dried <0.40 g H 2 O/g dry
weight and poor or lack of fertility when dried <0.10 g
H 2 O/g dry weight [42].
634
Anna Nebot et al.