RFLP analysis of genotypic variation in callus
--K. J. Bates and D. B. Walden

An RFLP analysis was conducted on maize DNA derived from six time-course 'windows': immature embryo (20 day), plumule (5 day), mature leaf, and callus aged 4 weeks, 12 weeks, and 18 weeks. The generation of callus tissue and the variation observed was reported in MNL (67:75,1993). Samples from genotypes A188, A632, B37, B73, CO159, F2, M14, Mo17, N28, Oh43, ONTARIO FLINT, VA26, W23, and W64A were digested with EcoRI, BamHI, HindIII, BstI, or EcoRI/BamHI and probed with a 14 kb EcoRI-waxy fragment, and 2 ubiquitin clones. Variation was observed for all waxy/enzyme combinations at each 'window'. Variations in fragment size and number, indicative of genotypic instability was observed particularly during the callus 'windows'. All data were generated using the DIG (digoxygenin) detection system, the method described by our lab in MNL (67:74, 1993). Genotypic RFLP grouping was observed at each 'window' for all enzyme/waxy combinations. This report identifies the differences observed for the genotypes for three restriction enzyme/waxy combinations at the immature embryo and callus (4, 12 and 18 week) 'windows'.

The most common restriction fragment pattern observed for EcoRI/waxy hybridizations of seven genotypes was a single fragment with an increase in fragment number at the 12 week callus stage. An increase in fragment number was observed only after 18 weeks of callus culture for Ontario Flint. In addition, a decrease in fragment size was observed in four of five genotypes after 18 weeks, however, A188 remained unchanged. Genotypic grouping in size RFLP was observed for DNA from all four 'windows' (immature embryo and callus at 4, 12 and 18 weeks). A trend towards increasing fragment size was observed for four of seven genotypes until the 12th week of callus, with a size decrease observed at the 18 week window. Deviations from this pattern included Ontario Flint, W64A and B73. An increase in fragment size was observed from the immature embryo 'window' to the first four weeks of callus only; fragment size stability was observed in the 12 and 18 week 'windows'. W64A also showed an increase in fragment size until the 12 week 'window', then stabilized. B73 exhibited the most deviant pattern, with a decrease in fragment size and number between the 4, 12 and 18 week callus windows.

RFLP's for seven genotypes were observed from HindIII/waxy hybrizations. Five of seven genotypes exhibited a single fragment for immature embryo and callus at 4 and 12 weeks, with an increase in fragment number at the 18 week 'window' for three of the five genotypes. B73 and B37 exhibited fragment number stability for all four stages. A decrease in fragment size was observed for three of seven genotypes. The smallest fragment observed was at the four week callus stage for B73. Both B73 and B37 exhibited an increasing size trend as callus aged. Oh43 exhibited a completely different, unstable size pattern, whereas A188 exhibited the most stability in fragment size, for the same fragment was observed in all four 'windows', even as a double restriction fragment pattern at 18 weeks. Overall, Ontario Flint exhibited the most variation in fragment size and in number when the immature embryo, and callus at 4, 12, and 18 week 'windows' were compared.

RFLP's for eight genotypes were observed from BamHI/waxy hybridizations. For five genotypes, an increase in fragment number at the 12 week callus phase was observed, with a decrease to a single fragment at the 18 week 'window'. A188 exhibited the most variation in fragment number, with single, double and triple restriction pattterns observed. A single fragment was observed for Ontario Flint at all 'windows' except the immature embryo stage, where a triple fragment pattern was found. All genotypes exhibited band sizes within a 3-5 kb size range, however, four of eight genotypes showed an increasing size trend as callus aged. Larger fragments up to 13 kb were observed at the 12 week stage for Ontario Flint, M14 and Oh43. Most changes observed amongst the genotypes and subsequent 'windows' involved fragment number alterations, with sizes remaining within the 3-5 kb range.

For HindIII and EcoRI digests, most double fragment patterns included a common sized band as well as one larger, > 30 kb fragment. These fragments were prevalent in DNA isolated after the 12 - 18 week callus phase.

Collectively, these data may give an indication of the loss of restriction sites due to either direct changes in the DNA, its packaging, or other factors interacting with the DNA after isolation. However, these patterns were observed for many genotypes and were particular in size, indicating a deliberate change, instead of a random process. In general, the most frequently observed EcoRI and BamHI fragment changes were at the 12 week callus window, whereas most HindIII RFLP's were observed after the 18th week of callus. Further research is required to address the question of whether these apparent changes in DNA during callus reproduction could be fore-runners of somaclonal variants. 


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