Inheritance of a mutable phenotype that is activated in alfalfa tissue culture

Mutable alleles, typically regulated by transposable elements, have been identified in a number of plant species. Tissue culture induced genomic shocks may activate such elements. A mutable recessive condition arose in tissue culture of alfalfa and was highly unstable in subsequent cycles of culture. The mutable allele, designated c2-m4, is allelic to the C2 locus, a basic color factor locus involved in anthocyanin synthesis. Current research has focused on the inheritance and instability of c2-m4 in new genetic backgrounds as well as on dosage effects of the allele. We have confirmed a previous report that c2-m4 reverts to function at much higher frequencies in vitro (reversion frequency ca. 0.23) than in planta (reversion frequency < 0.001). Over sexual generations c2-m4 continues to be unstable. Transmission of the mutable phenotype to selfed, testcross, and F2 populations demonstrated monofactorial inheritance patterns (P > 0.25). In populations expected to have some plants carrying two or more c2-m4 alleles, individuals were found that reverted in vitro at significantly higher frequencies than their parent (0.67 ± 0.04 and 0.44 ± 0.08 versus 0.20 ± 0.09). In planta reversion also increased with increasing c2-m4 dosage. Preliminary evidence indicates that as c2-m4 dosage increases, each allele maintains its original capacity to revert.Key words: somaclonal variation, transposable element, tissue culture, mutable allele.

Controlling vitrification in Larixdecidua via culture media manipulation

Vitrification rates obtained from our inverted embryo system were significantly decreased by lowering the cytokinin concentrations in Brown and Lawrence medium containing 10 mM L-glutamine from 10 to 1 mg/L, or by replacing 10 mM L-glutamine with equimolar concentrations of Ca(NO3)2 or by adding 1 g/L Gelrite to the normal 10 g/L Difco–Bacto agar in the culture media. In all treatments, decreased vitrification was accompanied with decreased adventitious bud production. With reduced N6-benzylaminopurine, vitrification decreased from 77 to 29%, but bud production decreased from 61 to 17 buds per explant and mortality increased from 3 to 33%. Incorporation of Ca(NO3)2 into the media decreased vitrification from 87 to 21%, but the number of adventitious buds per embryo decreased from 75 to 42. Vitreous shoots were reverted to normal development with an 81% reversion frequency and a 6% mortality rate by culturing these shoots on Gresshof and Doy medium with one-half total nitrogen. Elongation of previously vitreous shoots was best when these shoots were cultured on Gresshof and Doy medium + 0.5 mg/L N6-benzylaminopurine for 2 weeks, followed by subculture on Gresshof of and Doy medium + 10 g/L charcoal.

Activation of a nonexpressed hypoxanthine phosphoribosyltransferase allele in mutant H23 HeLa cells by agents that inhibit DNA methylation

HeLA H23 cells are a mutant female human tumor cell line harboring defective hypoxanthine phosphoribosyltransferase (HPRT; IMP-pyrophosphate phosphoribosyltransferase, EC 2.4.2.8) as a result of a mutation that alters the isoelectric point of the enzyme (G. Milman, E. Lee, G. S. Changas, J. R. McLaughlin, and J. George, Jr., Proc. Natl. Acad. Sci. USA 73:4589-4592, 1976). As shown by Milman et al. and confirmed by us here, rare HAT+ revertants arise spontaneously at 1.9 X 10(-8) frequency and express both mutant and wild-type polypeptides. Thus, the H23 mutant also carries a silent wild-type HPRT allele that is activated in revertants. To test whether the silent allele was activated via hypomethylation of genomic DNA, H23 cells were treated with inhibitors of DNA methylation, and revertants were scored by HAT or azaserine selection. At an optimal dose of 5 microM 5-azacytidine, the reversion frequency was increased about 50-fold when assayed by HAT selection and over 1,000-fold when assayed by azaserine selection. HAT+ and azaserine revertants were heterozygous for HPRT, expressing both wild-type and mutant HPRT polypeptides. Like spontaneous revertants, they contained active HPRT enzyme and were genetically unstable, reverting at about 10(-4) frequency. Similar results were found after treatment with N-methyl-N'-nitro-N-nitrosoguanidine, a DNA-alkylating agent and potent inhibitor of mammalian DNA methylation. By contrast, the DNA-ethylating agent, ethyl methanesulfonate (EMS), did not increase the HAT+ reversion frequency; it did, however, increase the frequency by which H23 revertants heterozygous for HPRT reverted to 6-thioguanine resistance. Of nine EMS revertants, seven lacked HPRT activity and had a substantially reduced expression of the wild-type polypeptide. These observations support the hypothesis that DNA methylation plays an important role in human X-chromosome inactivation and that EMS can inactivate gene expression by promoting enzymatic methylation of genomic DNA as found previously for the prolactin gene in GH3 rat pituitary tumor cells (R. D. Ivarie and J. A. Morris, Proc. Natl. Acad. Sci. USA 79:2967-2970, 1982; R. D. Ivarie, J. A. Morris, and J. A. Martial, Mol. Cell. Biol. 2:179-189, 1982).

Activation of a nonexpressed hypoxanthine phosphoribosyltransferase allele in mutant H23 HeLa cells by agents that inhibit DNA methylation.

HeLA H23 cells are a mutant female human tumor cell line harboring defective hypoxanthine phosphoribosyltransferase (HPRT; IMP-pyrophosphate phosphoribosyltransferase, EC 2.4.2.8) as a result of a mutation that alters the isoelectric point of the enzyme (G. Milman, E. Lee, G. S. Changas, J. R. McLaughlin, and J. George, Jr., Proc. Natl. Acad. Sci. USA 73:4589-4592, 1976). As shown by Milman et al. and confirmed by us here, rare HAT+ revertants arise spontaneously at 1.9 X 10(-8) frequency and express both mutant and wild-type polypeptides. Thus, the H23 mutant also carries a silent wild-type HPRT allele that is activated in revertants. To test whether the silent allele was activated via hypomethylation of genomic DNA, H23 cells were treated with inhibitors of DNA methylation, and revertants were scored by HAT or azaserine selection. At an optimal dose of 5 microM 5-azacytidine, the reversion frequency was increased about 50-fold when assayed by HAT selection and over 1,000-fold when assayed by azaserine selection. HAT+ and azaserine revertants were heterozygous for HPRT, expressing both wild-type and mutant HPRT polypeptides. Like spontaneous revertants, they contained active HPRT enzyme and were genetically unstable, reverting at about 10(-4) frequency. Similar results were found after treatment with N-methyl-N'-nitro-N-nitrosoguanidine, a DNA-alkylating agent and potent inhibitor of mammalian DNA methylation. By contrast, the DNA-ethylating agent, ethyl methanesulfonate (EMS), did not increase the HAT+ reversion frequency; it did, however, increase the frequency by which H23 revertants heterozygous for HPRT reverted to 6-thioguanine resistance. Of nine EMS revertants, seven lacked HPRT activity and had a substantially reduced expression of the wild-type polypeptide. These observations support the hypothesis that DNA methylation plays an important role in human X-chromosome inactivation and that EMS can inactivate gene expression by promoting enzymatic methylation of genomic DNA as found previously for the prolactin gene in GH3 rat pituitary tumor cells (R. D. Ivarie and J. A. Morris, Proc. Natl. Acad. Sci. USA 79:2967-2970, 1982; R. D. Ivarie, J. A. Morris, and J. A. Martial, Mol. Cell. Biol. 2:179-189, 1982).