1975; Brown et al. 1988; Ayabe and Sumi 2001). Apical meristems in plants are suitable
explants for the production of virus-free plants since the infected plant’s meristems typi-
cally harbor titers that are either nearly or totally virus-free. Meristem culture in combi-
nation with thermotherapy has resulted in successful production of virus-free plants
when meristem culture alone is not successful (Kartha 1986; Manganaris et al. 2003;
Wang et al. 2006).
5.7 Regeneration Methods of Plants in Culture
In plant biotechnology, tissue culture is most important for the regeneration of transgenic
plants from single transformed cells. It is safe to say that without tissue culture there
would be no transgenic plants (although this situation is slowly changing—nonetheless
tissue culture is required to regenerate intact plants in most species).
5.7.1 Organogenesis
Organogenesisis the formation of organs: either shoot or root. Organogenesis in vitro
depends on the balance of auxin and cytokinin and the ability of the tissue to respond to
phytohormones during culture. Organogenesis takes place in three phases. In the first
phase the cells become competent; next, they dedifferentiate. In the third phase, morpho-
genesis proceeds independently of the exogenous phytohormone (Sugiyama 1999).
Organogenesis in vitro can be of two types: direct and indirect.
5.7.1.1. Indirect Organogenesis.Formation of organs indirectly via a callus phase is
termedindirect organogenesis. Induction of plants using this technique does not ensure
clonal fidelity, but it could be an ideal system for selecting somaclonal variants of
desired characters and also for mass multiplication. Induction of plants via a callus phase
has been used for the production of transgenic plants in which (1) the callus is transformed
and plants regenerated or (2) the initial explant is transformed and callus and then shoots are
developed from the explant.
5.7.1.2. Direct Organogenesis. The production of direct buds or shoots from a
tissue with no intervening callus stage is termeddirect organogenesis(Fig. 5.10).
Plants have been propagated by direct organogenesis for improved multiplication
rates, production of transgenic plants, and—most importantly—for clonal propa-
gation. Typically, indirect organogenesis is more important for transgenic plant
production.
5.7.1.2.1. Axillary Bud Induction/Multiple-bud Initiation.This technique is the most
common means of micropropagation since it ensures the production of uniform planting
material without genetic variation. Axillary shoots are formed directly from preformed mer-
istems at nodes (Fig. 5.11), and chances of the organized shoot meristem undergoing
mutation are relatively low. This technique is often referred to asmultiple-bud induction.
Many economically important plants have been propagated using this method. Multiple-
bud initiation has been successful in crop plants but in only a few tree species such as
Millingtonia hortensis(Hegde and D’Souza 1995) andFagus sylvatica(Chalupa 1996).
5.7. REGENERATION METHODS OF PLANTS IN CULTURE 125