Proc. Nati. Acad. Sci. USA Vol. 91, pp. 5222-5226, June 1994 Review Genetic instability of plant tissue cultures: Breakdown of normal controls (DNA methylation/genome rearrangements/rearrangement induced premeioticafly/repeat-induced point mutation/heterochromatin) R. L. Phillips*t, S. M. Kaepplert, and P. Olhoft* *Deprtnent of Agronomy and Plant Genetics and Plant Molecular Genetics Institute, University of Minnesota, St. Paul, MN 55108; and *Department of Agronomy, University of Nebraska, Lincoln, NE 68583

ABSTRACT Plants regenerated from However, the frequency of various nated the cell or cells for the tissue cul- relatively undifferentiated callus cultures classes of mutants derived from plant ture. The many levels of genomic modi- possess a vast array of genetic changes. tissue culture is elevated far beyond that fication that already are known and ex- Such variations can result in useful agri- expected in nature. Clearly, tissue cul- pressed as changed genotypes and cultural and horticultural products. For ture processes that involve an undiffer- phenotypes could be potent sources for other purposes, however, variations in entiated callus phase are mutagenic. selection by the plant breeder, and inci- traits other than those of interest may be Variations include, but are not restricted dentally, for theoretical ponderings by undesirable-for example, using cultured to, chromosomal rearrangements and the biologist." cells for genetic engineering. Any steps single-gene mutants (mostly recessive). This article reviews the progress in made toward understanding the basis of DNA methylation changes also have understanding the basis of tissue culture- tissue culture-induced genetic variation been reported in regenerated plants and induced variation. It also explores possi- should be helpful in developing a more their progeny. ble links between tissue culture-induced stable and manipulatable somatic cell sys- In her Nobel lecture, Barbara Mc- variability and mechanisms of sequence tem. This review provides a glimpse at the Clintock (2) said "Some responses to or genomic change. specific kinds of genetic changes encoun- stress are especially significant for illus- Change in tissue culture most likely tered among regenerated plants and their trating how a genome may modify itself occurs by a stress-response mechanism. progeny. Included among these variations when confronted with unfamiliar condi- The relevant mechanism may best be are cytosine methylation alterations of the tions. Changes induced in genomes when described as a programmed loss of cellu- genome. The repeat-induced point muta- cells are removed from their normal lo- lar control. The most commonly ob- tion (RIP) phenomenon, reported for fil- cations and placed in tissue culture sur- served plant tissue culture-imposed amentous fungi, is invoked to provide a roundings are outstanding examples of changes-chromosome rearrangements, framework to consider the origin of vari- this. The establishment of a successful DNA methylation, and mutations-also ation in plant tissue cultures. tissue culture from animal cells, such as are salient features of a phenomenon those of rat or mouse, is accompanied by called repeat-induced point mutation Cells of all living organisms reproduce readily observed genomic restructuring. (RIP, formerly termed rearrangement- with almost exact fidelity and give rise to None of these animal tissue cultures has induced premeiotically) first described in daughter cells of predictable genotype. given rise to a new animal. Thus, the filamentous fungi (3, 4). This review will Errors in the process occur infrequently significance of these changes for the or- describe features of RIP and of tissue due to a remarkable variety of cellular ganism as a whole is not yet directly culture-induced variation. It will also dis- controls that regulate the genome. These testable. The ability to determine this is a cuss processes that might induce the RIP- controls cause expression of genes in a distinct advantage of plant tissue cul- like system in plant tissue culture. developmentally specific manner and fa- tures." cilitate the duplication, recombination, McClintock went on to say "The treat- A Review of Tissue Culture-Induced and distribution of chromosomes. Nor- ment, from isolation ofthe cell or cells of Variation in Plants mal cell behavior is the result of a com- a plant, to callus must inflict on the cells plex cascade of genetic programs that is a succession of traumatic experiences. Mutation induced by plant tissue culture sensitive to disruption by biotic and abi- Resetting of the genome, in these in- has been the subject of numerous scien- otic stresses (1). Although certain forms stances, may not follow the same orderly tific inquiries (reviewed in refs. 5-17). of mutagenesis result from a direct sub- sequence that occurs under natural con- Two general concepts have emerged stitution, deletion, or insertion of base ditions. Instead, the genome is abnor- from these studies. (i) Tissue culture- sequences, genetic changes also can be mally reprogrammed, or decidedly re- induced mutation has been detected in all "self-imposed" by a breakdown of the structured. These restructurings can give species studied. Rarely have individual normal cellular controls on chromo- rise to a wide range ofaltered phenotypic genotypes within species been identified somes. If the change deals with large expressions. Some of the altered pheno- which show no mutation, and in those domains of the genome, a variety of al- types are readily observed in the newly cases they most likely resulted from sam- terations in chromatin and in gene ex- produced plants themselves. Others ap- pling small numbers of individuals or pression can occur. pear in their progeny. Their association from scoring for only one or a few types The past 20 years of research on plant with genomic change remains proble- ofmutation. Trends toward low mutation tissue cultures, regenerated plants, and matic. Other altered phenotypes clearly rates within species are usually corre- progenies of the regenerated plants have reflect genomic restructuring, and vari- revealed a rich array of culture-induced ous levels of this have been observed. It Abbreviation: RIP, repeat-induced point mu- genetic variants. The genetic behavior of may be safe to state that no two of the tation. these variants generally appears similar callus-derived plants are exactly alike, tTo whom reprint requests should be ad- to that of naturally occurring mutants. and none is just like the plant that do- dressed. 5222 Downloaded by guest on September 28, 2021 Rmveview: Phillips et al. Proc. Natl. Acad. Sci. USA 91 (1994) 5223 lated with their "culturability." Cell single-gene phenotypic mutants, it arises cycle controls, which prevent cell divi- growth and regenerability indicate low at least as frequently. sion before the completion of DNA rep- levels of cell stress, levels which exper- Specific genetic changes associated lication, are presumed to be disrupted by imentally are reflected by relatively with particular tissue culture-induced tissue culture, resulting in chromosome lower mutation rates. (ii) Tissue culture- phenotypic mutants have been elucidated breakage. Chromosome breakage with- induced mutation is a general increase in only in rare cases (25, 26). However, a out the reunion ofbroken fragments leads most types of mutation rather than in a variety ofmutation types have been char- to deletions of chromosome segments; few specific types. Tissue culture- acterized which most likely are responsi- chromosome breakage followed by the induced variation has usually been based ble for the observed phenotypic variation. reunion of broken ends leads to translo- on phenotypic differences in regenerated These changes include cytological aberra- cations, inversions, duplications, and de- plants and their progeny. However, ge- tions which are primarily the result of letions. chromosome breakage, single base Chromosome breakage could be in- nomic changes appear to be the basis for altered levels of DNA the phenotypic alterations. changes, changes in the copy number of duced by methyl- repeated sequences, and alterations in ation. Heterochromatinization of chro- Phenotypic changes found in regener- matin has been associated with increased ated plants and their progeny are most DNA methylation pattern. The genomic changes represent a wide array of differ- methylation. Nucleosomes of mouse strikingly observed as qualitative mu- chromatin have been fractionated and tants which have major phenotypic ef- ent alterations. The thread connecting these diverse mutations is that all could be probed with antibodies against 5-methyl- fects (Table 1). The mutants have phe- cytosine (27). More methylcytosine is notypes similar to those previously ob- the result ofa disruption ofnormal cellular associated with isolated nucleosomes served in the respective species, such as controls. Pardue (1) has hypothesized that is not the default state that contain histone H1. Because H1 is chlorophyll deficiencies, dwarfs, and de- genomic stability involved in chromosome condensation, fective seeds. In some cases, allelism but is the result of a rather finely tuned increased methylcytosine conceivably with previously characterized mutants system ofchecks and balances. The tissue culture environment may cause a general could affect the rate of DNA replication. has been proven. The qualitative mutants disruption of cellular controls, leading to This delayed replication could cause are generally inherited as single Mende- the numerous genomic changes present in anaphase bridges, chromosome break- lian factors and are most frequently re- tissue culture regenerants. There are age, and rearrangements. The type of cessive alleles. The relative frequency many examples of observed genomic rearrangement would depend on the and gene action of the different types of changes to the cellular process which may chromosome location of the heterochro- qualitative mutations are consistent with be functioning abnormally. matin, whether homologues or hetero- studies using mutagenic agents that in- Chromosome aberrations are fre- logues are involved, and the ploidy level duce mutations at random sites such as quently found in plants regenerated from (28). Alternatively, chromosome break- ethyl methanesulfonate. Several unstable tissue culture. These aberrations, which age could be caused by decreases in or nontransmissible mutations have been are the result of chromosome breakage methylation. Decreases in methylation observed, indicating that transposable el- events, have been most well character- have been implicated in the nondisjunc- ements and/or epigenetic modification ized in maize and oat (12) but have also tion of chromosomes in both rye (ref. 29; may be the basis of some tissue culture- been observed in other species. Translo- methylation decreased by exposure to induced mutations (ref. 18; C. L. Arm- cations, inversions, deletions, and dupli- 5-azacytidine) and Neurospora (ref. 30; strong and R.L.P., unpublished observa- cations have all been detected. The methylation decreased because ofmutant tion). Transposable element activity has breakpoints of the various aberrations methyltransferase). According to these been detected in the progeny of regener- generally have been found either be- mechanisms, maintenance ofDNA meth- ated plants (19, 20), but a direct associ- tween distal heterochromatin and the ylation rather than DNA replication ation between an active transposable el- centromere or within centric heterochro- could be the cellular process which has ement and a phenotypic mutant has not matin. Late replication of heterochro- gone awry and given rise to chromosome yet been demonstrated. Quantitative trait matic blocks followed by chromosome breakage events. variation has also been observed by sev- bridges and breakage has been hypothe- Single base-pair changes have been de- eral researchers (21-24). While this type sized as the mechanism explaining the tected in the progeny of tissue culture of variant is somewhat more subtle than location of the breakpoints. Normal cell regenerants and have been shown to be the basis of two independent, tissue cul- Table 1. Types of single-gene visible mutants segregating in progeny of regenerated ture-induced alcohol dehydrogenase mu- maize plants tants in maize (25, 26). More often, the occurrence of single base-pair changes Chlorophyll deficiencies Seed characteristics has been inferred based on the presence of Virescent Defective kernel restriction fragment length polymor- Pale green Shrunken phisms detected by specific-sequence Luteus Sugary probes (31, 32). Single base-pair changes Striate Viviparous could theoretically result by two mecha- lojap nisms: deamination of cy- Albino Leaf morphology (i) methylated Crinkly tosine, resulting in a C -* T or G -- A Yellow green Zebra Wilted transition following mismatch repair; and stripe Adherent (epidermal cell fusions) (ii) loss of precision of the DNA replica- Unstable albino tion/repair machinery, resulting in transi- Reproductive structures tion Necrotic leaves Male or transversion types of base-pair Lower leaf necrosis sterility changes. Both ofthese mechanisms could Ramosa tassel be due in Necrotic leaf (lethal) Another color to a reduction the normal con- Necrotic leaf spots trols which maintain a standard level of sequence integity. It is possible that se- Stature quence changes involving a small number Dwarf of contiguous nucleotides could also be Semi-dwarf the footprint left as a result of excision of Downloaded by guest on September 28, 2021 5224 Review: Phiffips et al. Proc. Natl. Acad. Sci. USA 91 (1994) a transposable element. The frequency of the methylated cytosines, thus the term high and low copy sequences. Recent element activity in maize does not cur- repeat-induced point mutation (RIP). evidence from restriction fragment length rently support transposable-element exci- Neurospora has a rather low level of polymorphism (47, 48) indicates that the sion as a major effector of contiguous DNA methylation but it is not known maize genome has a high degree of dupli- base-pair changes, although the range of whether this is relevant to the RIP phe- cation. Over 70% of the single-copy rice types ofelements tested for and the extent nomenon in this species. The frequency probes that were hybridized to maize ge- of the testing have certainly not been of RIP was higher for cases where the nomic DNA detected duplications. These exhaustive enough to exclude the possi- introduced sequence was linked to the sequences are usually not susceptible to bility. endogenous sequence. This observation the RIP process in normal plants, thus Tandemly repeated sequences show indicated that RIP might depend on the indicating that the plant genome has increased levels of instability in tissue pairing or matching of like sequences. reached a state of equilibrium. Base culture with "clonal" regenerants con- From an evolutionary viewpoint, it has changes due to methylation can occur. taining variable copy numbers (33, 34). been speculated (49) that such a process Evidence in mammals (49) and in plants Variability in the copy number of tan- makes the sequences divergent in order (50) supports the idea that cytosines have demly repeated sequences is a conse- to be less likely to recombine and thus been converted to thymines over time and quence of various cellular stresses and leads to rearrangements. that C -+ T transitions occur much more has been found to be the basis of several The phenomenon of RIP originally was frequently than other base-pair changes in human genetic diseases (35). It is not shown to occur in the premeiotic stage in methylated regions. Spontaneous deami- unexpected, therefore, that repeated- the dikaryotic ascogenous hyphae ofNeu- nation of 5-methylcytosines is the likely sequence variation has been detected rospora before the nuclei fused to give rise explanation for these changes (51, 52). among tissue culture regenerants and to the zygote (42). Later, Pandit and However, the rate ofmutation is very low that this variation may be responsible for Russo (43) found reversible gene inacti- relative to that found in tissue culture, some of the observed phenotypic vari- vation in somatic tissue, which suggests suggesting that the potent RIP mechanism ability. Copy-number variability is most the possibility ofRIP occurring in somatic is not operating. Why then is the RIP likely effected by mitotic recombination. plant tissue cultures. Because of the oc- mechanism induced by tissue culture and Either interchromatid unequal crossing currence ofthese events in other than the not in normal plants? over or intrachromatid exchange of in- premeiotic cell, the term repeat-induced A second difference between tissue cul- verted repeats could result in the loss or point mutation was preferred. In Ascobo- ture-induced variation and RIP in Neuro- gain of genetic information. lus, the induced methylation also is coex- spora is that methylation and sequence DNA methylation patterns are fre- tensive with the length ofthe duplications changes in Neurospora are specific to an quently altered by the tissue culture envi- (44), but the process is reversible, mean- area homologous to an introduced se- ronment (17, 28, 31, 32, 36-40). Exami- ing that the repeat-induced methylated quence, whereas methylation changes and nation ofregenerated plants or their prog- cytosines are replaced by unmethylated mutations found in tissue culture regener- eny with methylation-sensitive restriction cytosines instead ofremaining methylated ants are found frequently at sequences enzymes has revealed both hyper- and or being deaminated to form thymine. scattered throughout the genome of the hypomethylation, depending on the re- Although both methylation and mutations same regenerated plant. This fact argues port. Several kinds of probes have been are observed in plant tissue cultures, we for induction of the RIP process by a employed, including those detecting sin- will use the term RIP because mutations genome-wide mechanism rather than by gle-copy and repeated sequences, as well are observed at high frequencies. specific sequence or chromosomal dupli- as cDNAs for known expressed genes and A mechanism similar to RIP is cer- cations which do occur in culture (14). random Pst I genomic sequences. Alter- tainly present in plants (reviewed in ref. What genome-level process could initiate ations in methylation in plant tissue cul- 45). Quiescent duplicated sequences, such wide-ranging changes? tures do not appear to be restricted to such as the transposable element Ac, are Finally, the methylation changes found specific kinds of DNA sequences. present throughout the genome ofmaize. among tissue culture regenerants are not In summary, the types ofgenomic vari- Paramutation of the R locus has been always the result oflarge increases in the ation observed in tissue culture are di- shown in specific maize crosses. Cosup- frequency of methylated cytosine as is verse. The parallels between the varia- pression of transgenes is common with found in Neurospora. Our results indi- tion induced by tissue culture and the methylation and altered gene expression cate that hypomethylation is the rule, but seemingly unrelated RIP phenomenon found in both the transgenic duplicate as we recognize that other researchers (37), are intriguing. These parallels may hold well as the original sequence (see ref. 46 even those using the same inbred maize the key to further understanding this for a recent review on cosuppression). line, find hypermethylation as well as complex process. While these examples suggest a compar- hypomethylation. Why this difference ison and methylation of duplicated se- exists is not clear. IfRIP causes a general Applying the RIP Hypothesis to Plant quences, characterization of ensuing increase in methylation, why would prog- Tissue Culture base-pair changes is not as thorough in eny of regenerated maize plants usually plants as in Neurospora. have less methylation when specific se- Selker (42) reported that a DNA se- Regenerants from plant tissue culture quences are compared between controls quence transformed into Neurospora led contain a high frequency of mutations, (plants from kernels borne on the same to the mass methylation of the cytosines many methylation changes, and chromo- ear from which embryos were explanted specifically within the limits of the se- some structure abnormalities. It is the for culture initiation) and regenerated quence of both the introduced copy and frequency and types of mutations that plants? The explanation might lie in the the endogenous copy. The cytosine lead us to speculate that a RIP-like mech- RIP mechanism (Fig. 1). Methylated cy- methylation was indiscriminant in the anism is involved in tissue culture- tosines are deaminated and are therefore sense that any cytosine could be methyl- induced mutagenesis. However, a num- converted to thymines. This creates mis- ated, not just those present as CpG di- ber of issues suggest that the process of matched T-G base pairs. According to nucleotides or CpNpG trinucleotides mutagenesis in tissue culture is not ex- Brown and Jirieny (53), working with (where N could be any other base) as is actly like RIP as described in Neurospora. simian cells, the thymine is often (92%) the usual case. Point mutations occur The first difference is that the plant ge- repaired to a cytosine; note that this frequently in the newly methylated se- nome is littered with duplications includ- process replaces methylated cytosines quences as the result of deamination of ing disperse and tandem repeats of both with unmethylated cytosines. About 4% Downloaded by guest on September 28, 2021 Review: PhiUips et al. Proc. Natl. Acad. Sci. USA 91 (1994) 5225 Cytosine methylation CCGG GGCC (cytosine- CmC GG Deamination CT GG -< Repair of > CC GG Replication methylation lost) G GmCC GGmCC Mismatch to G:C GGmCC A GG'CC No repair (2%) (90%) CC GG I Replication Repair of mismatch to A:T (8%)

CTGG GGmCC CT GG Maintenance GACC CC GG GAmCC Methylation (Point mutation) I 4 Maintenance Replication G GmCC Methylation C-C GG 4 (No change) G GmCC CTGG GAmCC CmC GG GACC CT GG (No change) (Point mutation) (Point mutation)* FIG. 1. Potential creation of mutations and non-methylated cytosines in plant tissue cultures by RIP. *, The methylated cytosine will not be maintained due to loss of CG symmetry. mismatches stay as such until replica- repeat-induced mutation, since compari- 16% to 40% in only 5 days; 2 pg/ml is a tion, which then results in a transition son of repeats is not directly involved. commonly used concentration of2,4-D in mutation. In the remaining 4%, the gua- Does the evidence support the fre- monocot tissue cultures. Other auxins nine is repaired to an adenine, again quency of newly generated duplications such as 1-naphthaleneacetic acid and in- resulting in a mutation. needed to subsequently observe such a doleacetic acid also caused increases in The duplicated sequences in Neuro- high frequency ofmethylation alterations DNA methylation. This known effect of spora become riddled with C -+ T transi- and apparent point mutations? The evi- 2,4-D makes it a possible inducer of RIP tions following the extensive cytosine dence for duplications is mostly from in plant tissue cultures. However, the methylation. Since mismatches are usu- cytological analysis of meiotic tissue. extrapolation of this effect to cultures ally repaired to the original base, this Duplications are usually recognized as other than carrot has not been shown. implies that an even greater frequency of heteromorphic bivalents at diakinesis or Preliminary analysis of the amount of the TUG mismatches were repaired back to metaphase I or the occurrence of a buckle methylation in maize tissue culture found C-G pairs. In maize, with a higher level of at pachynema in heterozygotes. In both similar levels in non-cultured plants com- methylcytosines, RIP would be expected cases, distinction between duplications pared with callus cultures (P.O., unpub- to produce a higher frequency of methyl- and deletions is difficult. Many of the lished data). More research is needed on cytosine cytosine changes. This high aberrations are probably duplications, as the cytosine methylation effects of hor- frequency of change could explain why judged from the absence of the degree of mones other than 2,4-D, other media maize regenerants homozygous for new sterility expected from deletions. Al- components, and other culture systems. DNA methylation patterns occur at such though heteromorphic pairs are detected Hormones may effect tissue culture high frequency; 17% of the DNA methyl- at an appreciable frequency (14), cyto- variation by causing a general increase in ation changes are apparently homozygous logical analysis most likely reveals only a methylation in most monocot cultures. in the original regenerant (17). Depending small fraction of the total. The occur- However, hormones found in the tissue on the number of CmCGG sites repre- rence of a multitude of cytologically un- culture medium, such as 2,4-D, may act sented in the probed region (using Hpa II detectable rearrangements is likely. by a quite different mechanism. The her- and Msp I isoschizomers), a relatively Thus, a sufficient number ofduplications bicidal mode ofaction of2,4-D is not well high frequency of altered sites might be could be generated in tissue cultures to understood but involves sub- possible, leading to higher than expected initiate a substantial amount ofRIP. Most apparently homozygosity frequencies. of these duplications might be small stantial general increases in transcription enough to have normal transmission and, (55). The events leading to the increased Induction of Tissue Culture-Imposed therefore, possibly be subject to RIP in transcription may alter the chromatin Mutagenesis and the RIP Mechanism subsequent generations. structure. These alterations could disrupt Although an argument can be made for the stability of the genome and result in A RIP mechanism might be induced in duplications in tissue cultures inducing the comparison of repeated sequences, tissue culture in three ways: (i) duplica- the RIP process, agents in the medium an event which does not normally occur. tions occurring in tissue culture could also might be the leading cause. LoSchi- Among the features of RIP is the con- initiate the process, (ii) an agent in the avo et al. (54) have shown that a common tinuing mutation over cell cycles and tissue culture medium could substantially component of the plant tissue culture generations. In Neurospora, the degree increase the general level of sequence medium, the hormone 2,4-diphenoxyace- ofmutation is a function ofthe number of methylation with ensuing changes follow- tic acid (2,4-D), causes a dramatic eleva- cell cycles. If RIP is occurring in plant ing a RIP-like process, or (iii) the genomic tion of cytosine methylation in plant tis- tissue cultures, the commonly observed balance which inhibits RIP in normal sue cultures. An increase in 2,4-D con- culture age effect might be expected. The plants could be disrupted. The second centration in carrot (Daucus carota) older plant tissue cultures would have mechanism would better be described as suspension cultures from 0.5 to 2 pg/ml undergone more cell divisions and per- methylation-induced mutation rather than raises the percent 5-methylcytosine from haps more RIP. This is consistent with Downloaded by guest on September 28, 2021 5226 Review: Phillips et al. Proc. Natl. Acad. Sci. USA 91 (1994) the increasing mutation frequency ob- 1. Pardue, M. L. (1991) Cell 66, 427-431. schke, V. M. (1990) in Progress in Plant served as culture age increases. 2. McClintock, B. (1984) Science 226, 792- Cellular and MolecularBiology, eds. Nij- D. P. Cummings (personal communi- 801. kamp, H. J. J., VanDerPlas, L. H. W. & cation) observed maize tassels 3. Selker, E. U. & Stevens, J. N. (1985) Van Aartrijk, J. (Kluwer, Dordrecht, The cases of Proc. Natl. Acad. Sci. USA 82, 8114- Netherlands), pp. 131-141. sectored for anther color in sixth- 8118. 29. Neves, N., Barao, A., Castilho, A., generation lines derived from regener- 4. Selker, E. U., Cambareri, E. B., Jensen, Silva, M., Morais, L., Carvalho, V. & ated plants. The genes for anther color B. C. & Haack, K. R. (1987) Cell 51, Viegas, W. (1992) Genome 35, 650-652. should have been homozygous, since re- 741-752. 30. Foss, H. M., Roberts, C. J., Claeys, generation was from an inbred line. Ap- 5. Sunderland, N. (1973) in Plant Tissue K. M. & Selker, E. U. (1993) Science pearance of variants at later generations and Cell Culture, ed. Street, H. E. 262, 1737-1741. would be expected on the basis of RIP. (Blackwell, Oxford), 2nd Ed., pp. 177- 31. Muller, E., Brown, P. T. H., Hartke, S. 205. & Ldrz, H. (1990) Theor. Appl. Genet. The process occurs until a certain point is 6. D'Amato, F. (1977) in Plant Cell, Tissue, 80, 673-679. reached which has been postulated to and Organ Culture, eds. Reinert, J. & 32. Brown, P. T. H., Gobel, E. & Ldrz, H. depend on the ability of the duplicated Bajaj, V. P. S. (Springer, New York), (1991) Theor. Appl. Genet. 81, 227-232. sequences to still recognize each other. pp. 343-357. 33. Landsmann, J. & Uhrig, H. (1985) Theor. Those duplicated sequences which are 7. D'Amato, F. (1985) CRC Crit. Rev. Plant Appl. Genet. 71, 500-505. not linked take longer in Neurospora to Sci. 3, 73-112. 34. Brettell, R. I. S., Pallotta, M. A., Gus- undergo RIP, presumably because of the 8. Bayliss, M. W. (1980) Int. Rev. Cytol., tafson, J. P. & Appels, R. (1986) Theor. lower frequency of sequence pairing or Suppl. 11A, 113-144. Appl. Genet. 71, 637-643. A an 9. Larkin, P. J. & Scowcroft, W. R. (1981) 35. Sutherland, G. R. & Richards, R. I. matching. sequence duplicated at Theor. Appl. Genet. 60, 197-214. (1994) Am. Sci. 82, 157-163. unlinked position lost l1O% of the G-C 10. Larkin, P. J. & Scowcroft, W. R. (1983) 36. Quemada, H., Roth, E. J. & Lark, K. G. pairs in one generation, whereas a closely in Genetic Engineering of Plants: An (1987) Plant Cell Rep. 6, 63-66. linked duplicated sequence lost about Agricultural Perspective, eds. Kosuge, 37. Brown, P. T. H. (1989) Genome 31, 717- halfofthe G-C pairs after two generations T., Meredith, C. P. & Holleander, A. 729. (56). Thus, it is possible that new muta- (Plenum, New York), pp. 289-314. 38. Brown, P. T. H., Yoneyama, K. & tions could be produced several genera- 11. Orton, T. J. (1984) Adv. Plant Pathol. 2, LUrz, H. (1989) Theor. Appl. Genet. 78, tions removed from the regenerated 153-189. 321-328. plant. In Neurospora, duplications can 12. Benzion, G., Phillips, R. L. & Rines, 39. Brown, P. T. H., Kyozuka, J. & H. W. (1986) in Cell Culture and Somatic Sukekiyo, Y. (1990) Mol. Gen. Genet. undergo further RIP even after six gen- Cell Genetics ofPlants, ed. Vasil, I. K. 223, 324-328. erations (41). The proportion of progeny (Academic, New York), Vol. 3, pp. 435- 40. Kaeppler, S. M. & Phillips, R. L. (1993) showing RIP decreased with generations. 448. Proc. Natl. Acad. Sci. USA 90, 8773- Again, linked duplications diverged more 13. Larkin, P. J. (1987) Iowa State J. Res. 8776. than independent ones. Interestingly, 61, 393-434. 41. Cambareri, E. B., Singer, M. J. & Selker, many of the duplicated sequences in 14. Lee, M. & Phillips, R. L. (1988) Annu. E. U. (1991) Genetics 127, 669-671. maize are unlinked; perhaps a compari- Rev. Plant Physiol. Plant Mol. Biol. 39, 42. Selker, E. U. (1990) Annu. Rev. Genet. son of linked and unlinked repeats in 413-437. 24, 579-613. maize regenerants would shed light on 15. Sun, Z. X. & Zheng, K. L. (1990) in 43. Pandit, N. N. & Russo, V. E. A. (1992) Biotechnology in Agriculture and For- Mol. Gen. Genet. 234, 412-422. the importance of RIP in tissue culture- estry, ed. Bajaj, Y. P. S. (Springer, Ber- 44. Barry, C., Faugeron, G. & Rossignol, induced variation in this species. lin), Vol. 3, pp. 288-325. J.-L. (1993) Proc. Natl. Acad. Sci. USA 16. Peschke, V. M. & Phillips, R. L. (1992) 90, 4557-4561. Summary Adv. Genet. 30, 41-75. 45. Matzke, M., Matzke, A. J. M. & Mittel- 17. Kaeppler, S. M. & Phillips, R. L. (1993) sten-Scheid, 0. (1994) in Homologous Specific genomic alterations associated In Vitro Cell Dev. Biol. 291, 125-130. Recombination and Gene Silencing in with tissue culture variation have been 18. Oono, K. (1985) Mol. Gen. Genet. 198, Plants, ed. Paszkowski, J. (Kluwer, Dor- well characterized, but the mechanism 377-384. drecht, The Netherlands), in press. leading to these changes is not well un- 19. Peschke, V. M. & Phillips, R. L. (1991) 46. Flavell, R. B. (1994) Proc. Natl. Acad. Theor. Appl. Genet. 81, 90-97. Sci. USA 91, 3490-34%. derstood. There are numerous parallels 20. Peschke, V. M., Phillips, R. L. & Gen- 47. Helentjaris, T. D., Weber, D. & Wright, between RIP in Neurospora and tissue genbach, B. G. (1987) Science 238, 804- S. (1988) Genetics 118, 353-363. culture-induced variation in plants. How- 807. 48. Ahn, S. & Tanksley, S. D. (1993) Proc. ever, differences in the two mechanisms 21. Baenziger, P. S., Wesenberg, D. M., Natl. Acad. Sci. USA 90, 7980-7984. also exist. Understanding why differ- Schaeffer, G. W., Galun, E. & Feldman, 49. Kricker, M. C., Drake, J. & Radman, M. ences and similarities exist in these two M. (1983) Proc. Int. Wheat Genet. Symp. (1992) Proc. Natl. Acad. Sci. USA 89, systems should ultimately lead to a better 6th, 575-581. 1075-1079. understanding of tissue culture-induced 22. Zehr, B. E., Williams, M. E., Duncan, 50. Hepburn, A. G., Belanger, F. G. & Mat- variation. It seems likely that a preexist- D. R. & Widholm, J. M. (1987) Can. J. theis, J. R. (1987) Dev. Genet. 8, 475- such Bot. 61, 491-499. 493. ing mechanism for genomic change, 23. Lee, M. L., Geadelmann, J. L. & Phil- 51. Lindahl, T. & Nyberg, B. (1974) Bio- as RIP, could be the effector of tissue lips, R. L. (1988) Theor. Appl. Genet. 75, chemistry 13, 3405-3410. culture-induced mutagenesis. Under- 841-849. 52. Bird, A. P. (1980) Nucleic Acids Res. 8, standing the mechanism of mutation will 24. Dahleen, L. S., Stuthman, D. D. & 1499-1504. lead to a better understanding of (i) ge- Rines, H. W. (1991) Crop Sci. 31, 90-94. 53. Brown, T. C. &Jirieny, J. (1987) Cell 54, nomic change in response to stress, (ii) 25. Brettell, R. I. S., Dennis, E. S., 705-711. factors contributing to genomic stability, Scowcroft, W. R. & Peacock, W. J. 54. LoSchiavo, F., Pitto, L., Giuliano, G., and (iii) methods to control variation (1986) Mol. Gen. Genet. 202, 335-344. Torti, G., Nuti-Ronchi, V., Marazziti, among tissue culture regenerants. 26. Dennis, E. S., Brettell, R. I. S. & Pea- D., Vergara, R., Orselli, S. & Terzi, M. cock, W. J. (1987) Mol. Gen. Genet. 210, (1989) Theor. Appl. Genet. 77, 325-331. This is paper no. 21,094, Scientific Journal 181-183. 55. Chen, L. G., Switzer, C. M. & Fletcher, Series, Minnesota Agricultural Experiment 27. Ball, D. J., Gross, D. S. & Garrard, R. A. (1972) Weed Sci. 20, 53-55. Station. This work was partly supported by W. T. (1983) Proc. Natl. Acad. Sci. USA 56. Cambareri, E. B., Jensen, B. C., Schab- U.S. Department of Agriculture Grant 91- 80, 5490-5494. tach, E. & Selker, E. U. (1989) Science 37301-6376. 28. Phillips, R. L., Kaeppler, S. M. & Pe- 224, 1571-1575. Downloaded by guest on September 28, 2021