hybrids as described below. Cell walls can be removed from explant tissue mechanically or
enzymatically; the latter is used most often. Enzymatic cell wall degradation was pioneered
by Cocking (1960). Ever since then, protoplast production has been applied to various crop
and tree species. Plant cell walls consist of cellulose, hemicellulose, and pectin, with lesser
amounts of protein and lipid (Dodds and Roberts 1995). Hence a mixture of enzymes is
necessary for degrading the cell wall. The enzymes that are commonly used are cellulase
and pectinase. Following enzyme treatment protoplasts are purified from cellular debris
by filtering using a mesh and then flotation on either sucrose or ficoll. They are cultured
in a high-osmoticum medium to avoid bursting. Protoplasts are cultured either on liquid
or solid medium. Protoplasts embedded in an alginate matrix and then cultured on solid
medium have better success rates of regeneration. The alginate provides cellular protection
against mechanical stress and gradients in environmental conditions during the critical first
few days of protoplast culture.
5.6.4.1. Somatic Hybridization.Protoplast fusion and somatic hybridization tech-
niques provide the opportunity for bypassing reproductive isolation barriers, thus facilitat-
ing gene flow between species. Fusion of protoplasts is accomplished by the use of PEG
[poly(ethylene glycol)]. Protoplast fusion has helped in the development of somatic
hybrids orcybrids(cytoplasmic hybrids). Protoplasts offer the possibility of efficient
and direct gene transfer to plant cells. DNA uptake has been found to be easier in proto-
plasts than into intact plant cells. Although protoplasts seem to be a very attractive means
for plant regeneration and gene transfer, they are very vulnerable to handling. One has to
be very careful when manipulating protoplasts. They have to be cultured on a medium
with a high osmoticum such as sucrose or mannitol; otherwise the protoplasts will
burst open, which is why plant regeneration from protoplasts has proven to be difficult.
Therefore, protoplasts are now used in cell culture studies mostly to study localization
of proteins and transient transgene assays.
5.6.5 Embryo Culture
Embryo cultureis a technique in which isolated embryos from immature ovules or seeds are
cultured in vitro. This technique has been employed as a useful tool for direct regeneration
in species where seeds are dormant, recalcitrant, or abort at early stages of development.
Embryo culture also finds use in the production of interspecific hybrids between inviable
crosses, whose seeds are traditionally condemned and discarded because of their inability
to germinate. In plant breeding programs, embryo culture goes hand in hand with in vitro
control of pollination and fertilization to ensure hybrid production. Besides this, immature
embryos can be used to produce embryogenic callus and somatic embryos (Ainsley and
Aryan 1998) or direct somatic embryos (Cardoza and D’Souza 2000).
5.6.6 Meristem Culture
In addition to being used as a tool for plant propagation, tissue culture is a tool for the
production of pathogen-free plants. Using apical meristem tips, it is possible to produce
disease-free plants. This technique is referred to as meristem culture,meristem tip
culture,orshoot tip culture, depending on the actual explant that is used. Although it
is possible to produce bacterium- or fungus-free plants, this method has more commonly
been used in the elimination of viruses in many species (Kartha and Gamborg
124 TISSUE CULTURE: THE MANIPULATION OF PLANT DEVELOPMENT