Mu-element modification was assessed by the failure of the restriction enzyme HinfI to cleave DNA at sites located within Mu-element terminal inverted repeats. Southern analysis of HinfI digests of DNA from H99/Mutator callus line 657-2B and various tissues of primary regenerants (R0) of this line revealed differences in Mu-element modification, with Mu elements in the immature cob of one R0 substantially undermethylated compared to those in husk tissue from the same plant. Primary regenerants of a bz-Mum8 callus line also showed tissue-specific modification differences, although these differences were slight. For example, a Mu-homologous restriction fragment of approximately 1.3 kb, probably representing an unmodified Mu element, was present in DNA from immature cob and young shoot tissues of two primary regenerants. This fragment was absent in DNA from leaf tissues of both plants; however, a novel higher-molecular-weight fragment was present in these leaf DNA's.
Tissue-specific differences in Mu-element modification have at least four possible explanations. First, it is possible that regenerated plants in which variation was seen were of multicellular origin. Second, uncharacterized tissue- or stage-specific factors could have been responsible for protecting at least some of the Mu elements from modification by general cellular methylases. Third, position effects could have influenced Mu-element methylation in certain tissues. If Mu elements were adjacent to regions of DNA that were hypomethylated in specific tissues, these elements could have remained hypomethylated as well. Fourth, the lower level of Mu-element modification seen in specific tissues could have been due to the immaturity of those tissues. A mathematical model which predicts an increase in genomic methylation with tissue maturity lends support to this argument (V. Walbot, personal communication).
Southern analysis was also used to compare DNA from various tissues of R1 plants (first-generation regenerant progeny of H99/Mu2 callus line 657-2B) that had been digested with restriction enzymes that cut outside Mu1 (EcoRI, HindIII, and XbaI). Three of ten R1 plants examined showed restriction fragment polymorphisms among tissues. In each of two plants derived from the same primary regenerant (88-141-1 and 88-176-6), restriction fragments which were present in DNA from immature cob tissue were absent in DNA from leaf or husk tissue. In one of these plants (88-176-6), the reverse was also true. In a third plant (88-140-7), derived from a different primary regenerant, a restriction fragment that was present in DNA from immature cob and husk tissues was missing in DNA from the leaf. In each of these cases, all restriction fragments seen in the R1 plants were also present in the parental R0 plants. Thus, the observed polymorphisms were considered indicative of the absence of one or more Mu elements in a particular tissue relative to other tissues.
It is unlikely that restriction fragment
differences among tissues can be traced to somaclonal variation. Although
it is known that tissue culture can induce chromosomal rearrangements and
deletions that could be reflected as altered restriction fragments, tissue-specific
differences such as these have not been reported. We are exploring the
possibility that these differences represent tissue-specific Mu-element
excisions. Further studies of both Mu-element modification changes
and restriction fragment polymorphisms among tissues are planned using
plants which have not gone through a tissue culture phase to help eliminate
either somaclonal variation or genetic chimerism as contributing factors.
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