7. After a growth period at 25/20
C for 2–4 weeks, most mint
species can be kept at low temperatures (2–10
C) and 12 h low
intensity light for longer storage intervals (1–2 years).
Temperature-sensitive genotypes can only be maintained at
25/20
C under a 16 h photoperiod (60 μmol/m
2 s) and
require sub-culturing every 6 months.
8. For garlic and shallots, isolation of single shoots from bunching
plants may result in growth retardation or even the death of
plantlets. Therefore, keep small bunches which perform better
in vitro.
9. Hyperhydration of shoots is a degenerative phenomenon
which describes the filling of the intercellular spaces of the
tissue with water giving the plant a glassy appearance. The
number of hyperhydrated or vitrified plantlets increases with
the age and the duration of the sub-cultivation. In garlic, the
phenomenon may be reduced by an increase of the ratio of
potassium nitrate to ammonium chloride reduces [21] and
cooling the bottom of the culture vessels. The subculture
period of cold-stored cultures should not exceed 1 year.
10. To control the efficacy of cryopreservation, rewarming of a
subsample of a statistical sufficient amount is always suggested
[22]. In general, and for each accession, one regenerated
explant is considered as secure. Therefore, an optimum
numeric ratio between the main sample and the regeneration
control is recommended [23] such as when 100 explants are
introduced into storage, 50 additional explants need to be
cryopreserved to test regeneration and regrowth after about
1 h in liquid nitrogen. When percentage of regeneration is
>30%, cryopreservation is successful. When percentage of
regeneration is between 10% and 30%, a second set of
150 explants, including 50 explants for control, is suggested
to be introduced to cryopreservation. When the average regeneration of both sets is below 10%, do consider to use different
donor material for cryopreservation. For safety reasons, divide
the samples into two to three equal sets and place them into
two tanks and/or store them at different locations.
11. Cultivation at constant temperatures is also possible, but the
percentage of regeneration might be lower.
12. Survival of the explants is characterized by yellowish green
color. Sometimes explants may swell or stop growing.
Regrowth is characterized by the development of regularly
formed shoots and later on roots.
Cryopreservation of Shoot Tips
659
C for 2–4 weeks, most mint
species can be kept at low temperatures (2–10
C) and 12 h low
intensity light for longer storage intervals (1–2 years).
Temperature-sensitive genotypes can only be maintained at
25/20
C under a 16 h photoperiod (60 μmol/m
2 s) and
require sub-culturing every 6 months.
8. For garlic and shallots, isolation of single shoots from bunching
plants may result in growth retardation or even the death of
plantlets. Therefore, keep small bunches which perform better
in vitro.
9. Hyperhydration of shoots is a degenerative phenomenon
which describes the filling of the intercellular spaces of the
tissue with water giving the plant a glassy appearance. The
number of hyperhydrated or vitrified plantlets increases with
the age and the duration of the sub-cultivation. In garlic, the
phenomenon may be reduced by an increase of the ratio of
potassium nitrate to ammonium chloride reduces [21] and
cooling the bottom of the culture vessels. The subculture
period of cold-stored cultures should not exceed 1 year.
10. To control the efficacy of cryopreservation, rewarming of a
subsample of a statistical sufficient amount is always suggested
[22]. In general, and for each accession, one regenerated
explant is considered as secure. Therefore, an optimum
numeric ratio between the main sample and the regeneration
control is recommended [23] such as when 100 explants are
introduced into storage, 50 additional explants need to be
cryopreserved to test regeneration and regrowth after about
1 h in liquid nitrogen. When percentage of regeneration is
>30%, cryopreservation is successful. When percentage of
regeneration is between 10% and 30%, a second set of
150 explants, including 50 explants for control, is suggested
to be introduced to cryopreservation. When the average regeneration of both sets is below 10%, do consider to use different
donor material for cryopreservation. For safety reasons, divide
the samples into two to three equal sets and place them into
two tanks and/or store them at different locations.
11. Cultivation at constant temperatures is also possible, but the
percentage of regeneration might be lower.
12. Survival of the explants is characterized by yellowish green
color. Sometimes explants may swell or stop growing.
Regrowth is characterized by the development of regularly
formed shoots and later on roots.
Cryopreservation of Shoot Tips
659