m.

l Storage and Cryopreservation

of in Vitro Cultures
Y.P.S. BAJA]1

1 General Account
Cells and tissue cultures are normally stored in a well-lighted room at 25°C (Fig. 1).
However, this requires periodic transfer to fresh media, involving not only man-power
and high costs, but also the hazards of contamination, and sometimes the loss of the
entire material/germplasm. Moreover, the cell cultures retained on periodic subcultures
undergo genetic erosion, such as change in ploidy level, mutations, endomitosis, trans-
location, gene amplification, etc. Thus, genetic stability of some of the useful soma-
clonal cell lines generated in vitro cannot be ensured. Another important aspect is the
conservation of germplasm. The existing germplasm banks for plants are customarily
supplied by seed collections. Although seeds offer an effective storage system for
genetic material, they are usually variable and the plants obtained from them do not
show the actual traits of the mother plants. At present, there are no reliable methods
for the long-term conservation of germplasm from vegetatively propagated crops and
those of plants with recalcitrant seed. Moreover, the germplasm of rare, elite, and
endangered plant species must be conserved. To circumvent these problems, in vitro
methods are being developed/refined, both for short- and long-term storage, and for the
conservation of germplasm (see Bajaj 1986a). Cryopreservation of in vitro cultures in
liquid nitrogen has enabled plant recovery in a number of species and has been recom-
mended for the long-term storage of germplasm, especially that of the vegetatively
propagated crops (Bajaj 1990a).

2 Nonfrozen Storage (Table 1, Figs. 1,2)

Various methods have been shown to reduce the growth rate of cultures and thus to
delay/reduce the subculture frequency. Some of these methods are:

1. Minimal Medium and Growth Retardants: The use of minimal medium and growth
retardants such as abscisic acid (Henshaw et al. 1978) or growing cultures in a
sucrose-free medium (Jones 1974) have helped to delay the subculture/transfer
period.

iFormer Professor of Tissue Culture, Punjab Agricultural University, Ludhiana, India.

(Present address: A-137 New Friends Colony, New Delhi 110065, India)

Biotechnology in Agriculture and Forestry, Vol. 17

High.Tech and Micropropagation I (ed. by Y.P.S. Bajaj)
ID Springer-Verlag Berlin Heidelberg 1991
362 Y.P.S. Bajaj

2. Mineral Oil Overlay: Caplin reported in 1959 that the mineral oil overlay of carrot
callus tissue considerably reduced the rate of growth and subsequently delayed the
transfer frequency. Recently, Augereau et al. (1986) stored tissue cultures of
various medicinal plants using mineral oil overlay for 4-6 months without subcul-
ture.
3. Desiccation: Nitzsche (1980) reported the growth of dried carrot callus after 1 year
of storage. Recently, Gray (1987; see also Chap. 111.2, this Vol.) observed that
somatic embryos of carrot, grape, and orchardgrass showed normal germination
after desiccation.
4. Low Pressure/Low Oxygen: Bridgen and Staby (1981) stored tissue cultures under
low atmospheric pressure and low oxygen.
5. Low Temperature Storage: The storage of cultures at nonfreezing temperatures
(2-8 0C) has been successfully applied to a large number of species (Table 1; see
also Aitken-Christie and Singh 1987).

Of all these methods, low temperature storage has been extensively used. The storage
of medicinal plant cell cultures at nonfreezing temperatures and its effect on secondary
metabolites has been reviewed (Hiraoka 1988). The in vitro plantlets of Chrysan-
themum (Fig. 1) and Petunia, mass propagated and stored at 4-5 °C for up to 6 years
(1978-1984), with occasional exposure to light in the culture room, flowered after
transfer to pots, and nc abnormalities were observed (Fig. 2).

Table L Various methods employed for the storage of tissue cultures (Bajaj 1986a)

Method of storage Plant species Culture References

1. Mineral oil overlay Dal/ells carota Callus Caplin (1959)

2. Desiccation Dal/cus carota Callus Nitzsche (1980)
3. Growth retardants Solanum spp. Shoot tip Henshaw et at. (1980)
4. Low atmospheric Chrysanthemum Callus/ Bridgen and Staby
pressure morifolium plantlet (1981)
5. Low temperature
9°C Vitis vinifera Shoot tip Galzy (1969)
1-4°C Fragaria sp. Meristem/ Mullin and Schlegel
plantlet (1976)
I-4°C Malus domestiea Buds/ Lundergen and Janick
shoots (1979)
2-4°C Lolium multiflorum Tiller buds Dale (1980)
2-6°C Medicago sativa Shoot tip Cheyne and Dale
(1980)
2-5°C Beta vulgaris Plantlets Miedema (1982)
2-4°C Pin liS radiata Shoots Smith (1985)
4-5°C Chrysanthemum Explants/ Bajaj (1986a)
morifolium plantlets
4-5°C Petunia hybrida Explants/ Bajaj (1986a)
plantlets
8°C Actinidia chinensis Shoot tip Monette (1986)