17.2—
Induced Variation and Selection
The spectrum of mutants recovered among cultured plant cells has so far been determined primarily by the growth characteristics and requirements of these cells in vitro. The strict prerequisite of a high minimum density for proliferation of cells (Street, 1973) or protoplasts (Nagata & Takebe, 1971) in vitro makes easier the selection for mutants resistant to antibiotics and antimetabolites than the selection for auxotrophs. The isolation of auxotrophs requires an experimental system composed of haploid cells in which the wild type cells are at a disadvantage compared to the mutants. This approach was used by Carlson (1970) to isolate six auxotrophic mutants of Nicotiana tabacum. The selective system was adapted from a procedure used successfully with mammalian cells (Puck & Kao, 1967). In a minimal medium containing 5-bromodeoxyuridine (BUdR), actively dividing prototrophic cells will incorporate more of this lightsensitive nucleoside analogue into DNA than will arrested auxotrophs. The auxotrophs will survive subsequent illumination at a much higher frequency than the prototrophs and will form clones on a supplemented medium. The mutant tobacco calluses which were isolated grew slowly on a minimal medium. Plants were differentiated from four of the mutant clones and these also grew slowly without supplementation. In genetic analyses of these four regenerated plants, three plants transmitted the mutant phenotype as a single recessive Mendelian factor and one displayed a more complex pattern of inheritance. The recovery of only leaky mutants may be due to a lack of functional diploidization of the N. tabacum genome. Haploid cells obtained from this amphiploid species may contain two copies of many metabolically essential genes and thus two mutational events may be required to delete completely certain functions (Carlson, 1970).
Cell lines which exhibit increased resistance to a wide variety of antimetabolites have been isolated and studied. Haploid cultures of petunia (Binding et al., 1970; Binding, 1972) and tobacco (Maliga et al., 1973) resistant to streptomycin have been selected. In genetic crosses involving a diploid plant obtained from one mutant tobacco clone, drug resistance segregated as a maternally inherited character (Maliga et al., 1973; Sz.-Breznovits et al., 1974).
Plant cell lines containing dominant or semidominant genetic markers may prove useful in selecting hybrid cells formed by protoplast fusion. This realization has motivated the isolation of cell lines resistant to 8-azaguanine and BUdR since such mutants are employed in selecting hybrids in mammalian cell systems. Resistance to these analogue precursors of nucleic acids in cultured mammalian cells may result from deficiencies in hypoxanthine:guanosine phosphoribosyl transferase (HGPRT) and thymidine kinase activities, respectively, or in the uptake of these analogues (Kit et al., 1963; Littlefield, 1964a; Breslow & Goldsby, 1969). In the presence of aminopterin, endogenous biosynthesis of thymidine and hypoxanthine is inhibited and cultured mammalian cells are dependent upon an exogenous supply of these substances (Fig. 17.1). As each
Figure 17.1
Simplified scheme showing pathways for synthesis of guanylate and thymidylate.
Endogenous biosynthetic steps blocked by aminopterin-inhibition of tetrahydrofolate
synthesis are shown as well as the genetic blocks which confer resistance to BUdR and 8-azaguanine.
mutant cell line is unable to utilize one of these compounds, only complementing hybrids formed by cell fusion survive in a medium containing hypoxanthine, aminopterin, and thymidine (the HAT system) (Littlefield, 1964b). Although a low concentration of aminopterin inhibits growth of sycamore cells (Bright & Northcote, 1974) it should not necessarily be assumed that this effect is caused by the same mechanism in both animal and plant systems. The isolation of cell lines of tobacco (Lescure, 1973) and sycamore (Bright & Northcote, 1975) resistant to 8-azaguanine has been reported. The resistance phenotype is not completely stable in the sycamore cell line in which HGPRT activi ty is half that of the wild type level. The latter result is not unexpected in a diploid system and emphasizes the necessity of using haploid cell lines in order to recover mutants with an anticipated recessive phenotype.
Resistance to BUdR has been selected in haploid tobacco (Maliga et al., 1973) and in presumably diploid sycamore (Bright & Northcote, 1974) and soybean (Ohyama, 1974) cells. The resistant sycamore and soybean cultures did not differ significantly from the wild type in uptake of thymidine or in the level of thymidine kinase activity (Bright & Northcote, 1974; Ohyama, 1974) and the basis for the resistance phenotype remains unknown. Marton and Maliga (1974) have observed transmission of BUdR resistance to progeny of plants derived from one mutant tobacco callus and unimpaired uptake and incorporation of thymidine in four resistant cell lines.
Heimer and Filner (1970) have isolated a cell line of tobacco which is insensitive to inhibition by threonine when grown in a medium containing nitrate as sole nitrogen source. By studying nitrate accumulation in this mutant under conditions in which nitrate reductase is chemically inactivated and in which nitrate uptake by wild type cells is repressed, it was concluded that regulation of nitrate uptake is altered in the resistant cell line. Such mutants should prove extremely useful in the investigation of nitrogen metabolism.
The potential agronomic importance of tissue culture techniques is illustrated by experiments in which screening of cultured somatic cells resulted in the recovery of mutants of N. tabacum resistant to wildfire disease (Carlson,
1973a). This disease of tobacco is caused by a bacterial pathogen, Pseudomonas tabaci, which produces a toxin structurally related to methionine (Stewart, 1971; Fig. 17.2). Populations of mutagenized haploid cells were plated in a medium containing an inhibitory concentration of methionine sulphoximine, an analogue of the wildfire toxin (Braun, 1955) and resistant clones were selected. Three homozygous diploid plants which were regenerated from these methionine sulphoximine-resistant calluses are less susceptible than the parent plant to the pathogenic effects of bacterial infection. One mutant plant contains wild type intracellular levels of free methionine and resistance appears to be genetically complex. In the other two resistant plants the endogenous concentration of free methionine is five times higher than in the wild type while the free pool sizes of other amino acids remain unchanged (Carlson, 1973a). The levels of free methionine in the heterozygous plants are intermediate between those in the wild type and in the homozygous mutant (Chaleff & Carlson, 1975a).
Figure 17.2
Structures of methionine sulphoximine,
wildfire toxin (Stewart, 1971), and methionine.
Intermediate methionine levels are consistent with the observed transmission of the resistance phenotype of these two plants as a single semidominant locus (Carlson, 1973a). These experiments suggest that selection for toxin resistance in vitro may provide a generalized procedure for generating disease resistant varieties. It is also evident that regulation of amino acid metabolism may be altered in cells selected for resistance to an amino acid analogue and that this mutational event may be expressed stably in mature plants derived from the mutant callus tissue. Additional support for this conclusion is provided by other research.
Widholm (1972a,b) has isolated presumably diploid cell lines of N. tabacum and Daucus carota which are capable of growth in the presence of a normally inhibitory concentration of 5-methyltryptophan. Crude extracts of the resistant cell lines (5-mtr ) contain a species of anthranilate synthetase which is less sensitive to feedback inhibition by tryptophan and 5-methyltryptophan than is the wild type enzyme. Endogenous levels of free tryptophan in 5-mtr cell lines of tobacco and carrot are 15 times and 27 times higher, respectively, than the wild
type levels. Plants could not be regenerated from the tobacco and carrot cells which synthesized a feedback-insensitive form of anthranilate synthetase. However, plants were obtained from analogue-resistant carrot cultures in which anthranilate synthetase activity was indistinguishable from the wild type and uptake of 5-methyltryptophan and tryptophan was reduced. Although no genetic studies with these plants have been reported, cell lines re-initiated from the plants maintained the same degree of resistance to 5-methyltryptophan as the original selected lines. Cell cultures of tobacco and carrot resistant to p -fluorophenylalanine were isolated also. Resistance to this analogue apparently resulted from diminished rates of transport and incorporation (Widholm, 1974).
Two attempts have been made to employ analogue resistance as a selective screen in obtaining callus cultures of cereals which overproduce amino acids essential to human nutrition. Chaleff and Carlson (1975b) recovered three cell lines of rice which were stably resistant to the lysine analogue S-2-aminoethylcysteine. Analyses of both free pool and total amino acid compositions showed that the resistant cultures contained elevated levels of several amino acids, including lysine, methionine, leucine and isoleucine. Cheng (1975) has selected cultures of Hordeum vulgare which are resistant to the same analogue and in which lysine levels were increased, as was the rate of incorporation of exogenously supplied lysine. Unfortunately, attempts to differentiate plantlets from the variant calluses were unsuccessful in both cases. Although much valuable information is to be obtained from research with variant cell lines, such studies are severely limited by the inability to examine genetic expression and transmission in the mature plant. Future investigations should attempt to utilize experimental systems in which plants may be differentiated from the altered cell lines.