Variation detected in in vitro regenerated plantlets, derived from somatic cell culture, is somaclonal variation and variant clones recovered are called somaclones (Evans et al. 1 984; Ahuja 1 987). Another type of variation observed in culture is gametoclonal variation, arising from the culture of haploid gametic tissue. Both somaclonal and gametoclonal variation can result from either pre-existing genetic heterogeneity of the explant source, or from changes caused by
in
vitro-induced stress (Muller et al. 1 990; Ogura 1 990; Cecchini et al. 1 992).4.4.1 Somaclonal variation in micropropagated plant species
Tissue-cultured plantlets may be regenerated either directly through organ culture (bud or nodal culture) or indirectly via a callus phase (adventitious embryogenesis or bud formation) (Cassells 1 985). Somaclonal variation occurs at higher frequencies in long term callus cultures or suspension cultures derived from callus, than in the more stable organ cultures (Ahuja 1 987). Experimental evidence suggests that variation in in vitro cultured plantlets is a result of aberrations existing in the donor tissue, plus variation induced by culture conditions (Evans et al. 1 984; Parfitt & Arulsekar 1 987; Patel & Thorpe 1 987).
Armstrong and Phillips ( 1 988) record the frequency of variation in maize regenerants to be greater (37.2%) in friable embryogenic callus than in organogenic cultures (24.2%). However, Chowdhury and Vasil ( 1 993) were unable to detect any DNA variation in sugarcane plantlets regenerated through an embryogenic pathway. Differences in these findings may be attributed to the genotype of the explant or to differences in culture
conditions. Normal frequencies of somaclonal variation are between 1-3% per regeneration cycle (Skirvin et al. 1 993).
Genetic variants are reported for a range of plant characteristics including morphological, physiological and chromosomal variation (Partitt & Arulsekar 1 987; Reuther 1 990b). Evaluation of initial in vitro regenerants (Ro) and their offspring (R1) reveal that the following genetic changes occur during culture: changes in chromosome number and structure (Evans et al. 1 984; Geier 1 99 1 ) ; single gene mutations in the nucleus (Ahloowali 1 986); changes in chloroplast and mitochondrial DNA (Day & Ellis 1 985; Evans et al. 1 984); and, mitotic crossing over and activation of transposable elements (Peschke et al. 1 987; Geier 1 99 1 ).
Initiation of plant tissue into in vitro culture imposes stress to the explant, causing restructuring of the genome in some plants that can give rise to the altered phenotypes discussed in this section. Altered phenotypes are observed in some plants regenerated from culture while others are lethal and do not survive (McClintock 1 984). Somaclonal variation has been reported in a number of micropropagated plant species (Heinz & Mee 1 97 1 ; Swartz 1 989; Davies 1 986; Geier 1 98 8 ; Reuther 1 990b; Isabel et al. 1 996), and has been reviewed extensively (Chaleff 1983; Scowcroft 1 98 5 ; Ahuja 1 987; Karp 1 989; Semal & Lepoivre 1 990).
4.4.2 Factors influencing in vitro genetic variation
Several factors affect genetic stability of in vitro-cultured plantlets including the species, genotype and ploidy level; age of the plant and the type and source of explant; composition of medium and cultural conditions; length of time in culture, i.e. duration of callus phase; frequency of transfer; growth pattern; and, mode of regeneration (Ahuja
Chapter 4 Genetic analysis: literature review 93
4.4.2.1 Ploidy level and genotype
The ploidy level and genotype of the starting material are important factors determining the extent of variability in culture (Geier 1 99 1 ). There is evidence that ploidy levels lower than normal are unstable. For example, somaclonal variation is highest in mono-haploid, lower in di-haploid and lowest in tetra-haploid potato genotypes. In contrast, when variation is compared between polyploid and diploid genotypes, polyploid species are found to be more tolerant to chromosome variation. Likewise, evidence also shows that there is higher variability in interspecific hybrids compared with their parental species (Swartz 1 99 1 ).
4.4.2.2 Explant source and age
Pre-existing genetic variation within the explant may contribute to variability in
in vitro
developed callus and regenerated plantlets. Explant age is critical and explants, such as meristematic regions or very young tissue, with a low potential for variation, are recommended (Swartz 1 99 1 ). Normally, regeneration from tissue explants results in a lower percent somaclonal variation than regeneration from protoplasts of the same initial material (George & Sherrington 1 984; Geier 1 99 1 ).4.4.2.3 Culture environment
The effect of medium supplements on the genetic stability of cultured tissue are often conflicting and not easy to construe. Plant growth regulators such as auxin and cytokinins may enhance chromosome variability by disrupting mitosis (Geier 1 99 1 ). For example, 2,4-D stimulates DNA synthesis and endoreplication which can lead to nuclear fragmentation (Swartz 1 99 1 ), and has also been implicated where increased mitotic crossing-over occurs. Genetic changes caused by 2,4-D can occur at any time during the culture process as such changes are linked directly with normal growth and cell division. Other factors such as insufficient nucleic acid precursors, the use of some antibiotics, high
94
concentrations of inorganic nitrates, and the accumulation of ethylene in culture vessels have the potential to induce mutations in culture (Swartz 1 99 1 ).
4.4.2.4 Length of culture period
The length of time between subcultures and the number of subcultures affects the genetic stability of in vitro cultures. Generally, variability increases with increased number of subcultures and time in culture (Geier 1 99 1 ; Swartz 1 99 1 ; Cecchini et a1. l 992). In some studies, genetic stability appeared to be unaffected by the number of subcultures (Swartz 1 99 1). However, there is a general trend towards increased genetic variability with increasing time in culture. Kumar and Walton ( 1 992) show that aging PI hybrid callus cultures of (Elymus canadensis x E. trachycaulus) increased the frequency of changes in ploidy level of regenerated plants. Variation in chromosome number was noted in 1 2% of regenerants. Likewise, Armstrong and Phillips ( 1 988) observed that the frequency of chimerism in regenerants, derived from embryogenic and organogenic maize cultures, increased with increasing culture age. Using molecular techniques, Muller et al. ( 1 990) demonstrate that lower levels of DNA polymorphisms could be detected in rice plantlets regenerated from callus incubated for 28 days than among those incubated for 67 days. It is not clear if the increased frequency of somaclonal variation, due to the length of the callus growth phase, is a direct effect of the maintenance of a proliferative callus phase or due to prolonged exposure to growth regulators such as 2,4-D, or due to a synergistic effect of both factors (Millier et al. 1 990; Geier 1 99 1 ; Swartz 1 99 1 ) .
4.4.2.5 Pattern of growth and mode of regeneration
In vitro developed plantlets regenerated via a callus phase typically have a higher percent of variability than those derived through organ cultures. Histological evidence does not support this, particularly in monocotyledons (Geier 1 99 1 ) . In micropropagated monocotyledons, e.g. asparagus, in addition to disorganized cell development, a type of callus frequently occurs similar to a mass of organ initials. These tissues undergo a pattern of cell division like meristems in intact plants and maintain their genetic stability when
Chapter 4 Genetic analysis: literature review 95 cultured. This theory has been proposed to explain the relative stability of monocotyledonous regenerants like lily, anthurium and asparagus (Geier 1 99 1).
Different types of callus tissue may develop from a single explant source (George & Sherrington 1984). Therefore, in addition to the 'meristematic-like' callus, other less morphogenic friable callus may develop that normally exhibit a high degree of somatic variability. Experimental evidence indicates that for some monocotyledonous species, embryogenic cultures, often derived from morphogenic callus, are more stable than organogenic cultures (Swedlund & Vasi1 1 985). Similarly, Chowdhury and Vasil ( 1 993) used restriction fragment length polymorphism (RFLP) techniques to show that sugarcane mitochondrial DNA remained unchanged during somatic embryo culture. Their investigation examined plantlets regenerated from calli, cell suspensions, cryopreserved cell suspensions and protoplasts. The lack of genetic variation may either be a result of mitochondrial DNA being unaffected by in vitro culture conditions or the selection of the embryogenic process for the regeneration of genetically normal cells. However, Armstrong and Phillips ( 1988) show that for some species, for example maize, this degree of genetic stability is not the case, finding friable, embryogenic maize cultures to be naturally more cytologically unstable than organogenic cultures.