IN VITRO CULTURE FOR MUTANT DEVELOPMENT

Abstract

The success of any in vitro mutagenesis programme depends on the establishment of reproducible in vitro plant regeneration procedures, optimization of mutagenic treatments, and efficient screening of the mutagenized populations for desired variations (Ahloowalia, 1998; van Harten et al., 1998; Jain, 2000, 2006, 2007). Cell and tissue culture techniques can improve effectiveness of mutation induction in several aspects. One of the major advantages of tissue culture is that a large number of regenerated plants can be handled at ease for mutagenic treatments (Jain, 2000; Predrieri, 2001). They offer a wide choice of plant material for mutagen treatment (in vitro axillary buds, organs, tissues, protoplasts, and cells) (Broertjes and van Harten, 1988; Constabel and Shyluk, 1994; Jain and Newton, 1989; Duncan, 1997) that is more suited to mutation induction techniques as compared to in vivo buds (Predrieri, 2001). The tissue structures from which plants originate are either multicellular or of single cell origin (Williams and Maheshwaran, 1986; Ochatt et al., 2005; Kulkarni et al., 2007). Normally chimeras are major problems in regenerated plants by mutagen treatment of multicellular structures such as shoot tips or axillary buds. By in vitro culture chimeras can easily be dissociated by several time subcultures of shoot cultures, say 4 generations M1V4 (Jain, 2000; Predrieri, 2001) This, as compared to in vivo buds, means less risk of obtaining chimera plants and a higher probability for mutated cells to express the mutation in the phenotype (Broertjes and van Harten, 1988). It also facilitates rapid completion of the propagation cycles of subculture aimed to separate mutated from non-mutated sectors (Ahloowalia, 1998). In vitro selection and cloning of selected mutants can also be effective when supported by adequate early evaluation tools (Ball, 1990; Duncan, 1997; Ochatt, 2006; Ochatt et al., 2005) as well large scale multiplication of mutant plants. Micropropagation is an ideal system for true-to-type multiplication of mutant plants by using shoot tips or axillary buds (Ahloowalia, 1998). Furthermore, tissue culture provides high phytosanitary conditions, which is an ideal system for obtaining healthy starting material and is maintained throughout the plant regeneration process (Ochatt et al., 1990; Cassels, 1998; Predrieri, 2001; Kulkarni et al., 2007). Therefore, in vitro mutagenesis should always be carried out on healthy explants. Otherwise endogenous contamination may compromise obtaining mutations, as well as selection and/or the survival of mutants.