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a Dep. of Plant and Microbial Biology, Univ. of California, Berkeley, CA 94720 USA
mjcho{at}nature.berkeley.edu
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMaterials and methodsResultsDiscussionREFERENCES |
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Abbreviations: BAP, 6-benzylaminopurine • 2,4-D, 2,4-dichlorophenoxyacetic acid • IE, immature embryo
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMaterials and methodsResultsDiscussionREFERENCES |
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In recent years, major cereal crop plants, once considered recalcitrant to genetic engineering approaches, have been successfully transformed by different approaches and explants (reviewed by Vasil, 1994). Most of these published transformation methods involve a period of in vitro culture, but only limited data are available on chromosomal aberrations in transgenic plants, except for tobacco (Nicotiana tabacum L.) (Matzke et al., 1994) and soybean [Glycine max (L.) Merr.] (Singh et al., 1998). Barley, a major cereal crop used as feed, malt, and food, is a target for genetic engineering. Several recent reports described successful stable transformation using in vitro-cultured barley tissue (Jähne et al., 1994; Ritala et al., 1994; Wan and Lemaux, 1994; Funatsuki et al., 1995; Hagio et al., 1995; Salmenkallio-Marttila et al., 1995; Koprek et al., 1996; Lemaux et al., 1996; Tingay et al., 1997; Cho et al., 1998, 1999; Zhang et al., 1999).
Because of the obligatory in vitro-culture phase of these transformation protocols and the seeming propensity of barley tissue to incur somaclonal variation (Bregitzer and Poulson, 1995; Bregitzer et al., 1998), the potential negative effects of chromosomal aberrations are particularly problematic for molecular approaches to barley improvement (Lemaux et al., 1999). Despite these potential negative effects, no detailed chromosomal analyses have been conducted on transgenic barley plants. In this paper, we investigate the cytological variation of transgenic plants compared with nontransgenic callus cells and plants regenerated from them and the potential correlation between phenotypic variation and changes in ploidy level.
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Materials and methods |
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TOPABSTRACTINTRODUCTIONMaterials and methodsResultsDiscussionREFERENCES |
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Callus Induction
Donor plants were grown under controlled conditions, i.e., humidity, temperature, lighting, in growth chambers as described (Wan and Lemaux, 1994). Immature embryos (IEs) of about 1.0 to 2.5 mm were isolated intact under a stereo dissecting microscope from spikes surface sterilized for 10 min in 20% (v/v) bleach (5.25% sodium hypochlorite) followed by 3 washes in sterile water. IEs, scutellum side down, were placed on DM medium (see below) in the dark at 24 ± 1°C for callus induction (Wan and Lemaux, 1994). Five to 7 d after initiation, germinating shoots and roots were removed by manual excision. Callus was randomly selected with no subculture for chromosome analysis at 0, 1, 3, 7, and 14 d on 149, 115, 109, 41, and 80 cells, respectively. The remaining callus was randomly selected and maintained on the same medium in the dark, subculturing at 3- to 4-wk intervals. After 3- to 5-d of incubation on fresh medium, 81, 65, and 135 cells from callus tissues at 4, 12, and 20 wk after initial callus induction were used for chromosome analysis, respectively. This culturing system is the same as the first transformation scheme described below except for the bialaphos selection used during transformation.
Production of Nontransgenic Plants
Nontransgenic plants were obtained from calli initiated from IEs and grown on each of five different MS (Murashige and Skoog, 1962)-based callus-induction media: (i) D medium containing 2.5 mg L-1 2,4-D and 0.1 µM CuSO4 (Cho et al., 1998), (ii) DM medium containing 2.5 mg L-1 dicamba and 0.1 µM CuSO4 (Wan and Lemaux, 1994), (iii) DC medium containing 2.5 mg L-1 2,4-D and 5.0 µM CuSO4 (Cho et al., 1998), (iv) DBC1 medium containing 2.5 mg L-1 2,4-D, 0.01 mg L-1 BAP and 5.0 µM CuSO4 (Cho et al., 1998), and (v) DBC2 medium containing 2.5 mg L-1 2,4-D, 0.1 mg L-1 BAP and 5.0 µM CuSO4 (Cho et al., 1998). Three-month-old calli initiated and maintained on D or DM medium were used directly for regeneration on FHG medium (Hunter, 1988). Highly regenerative tissues initiated and maintained on DBC1 or DBC2 medium under dim light conditions (approximately 10 to 30 µmol m-2 s-1, 16 h light) were also transferred onto FHG regeneration medium. Highly regenerative tissues are green or light green tissues which are converted from an embryogenic state into a state that more closely resembles the morphology of a shoot meristem culture (Cho et al., 1998; Lemaux et al., 1999). Callus tissues from DC medium were transferred to FHG medium (Hunter, 1988) for regeneration after an intermediate culturing step on DBC2 for 1 month to mimic conditions used for transgenic tissues. Regenerated shoots were transferred into Magenta boxes (Magenta Corp., Chicago) with rooting medium (callus-induction medium without phytohormones). Plantlets were transferred to soil in the greenhouse and roots from young plants were used for cytological analysis.
Production of Transgenic Plants
Transgenic plants were obtained via microprojectile bombardment as previously reported (Wan and Lemaux, 1994; Lemaux et al., 1996) or with modifications (Cho et al., 1998) using a mixture of two plasmids: (i) pAHC20 (Christensen and Quail, 1996) containing the herbicide resistance gene, bar, from Streptomyces hygroscopicus under the control of the maize ubiquitin Ubi1 promoter and first intron and followed by the 3'-untranslated region of nos and (ii) one of the following, p16 (Sørensen et al., 1996), pD11-Hor3 (Sørensen et al., 1996), pdBhGN1-2 (M.-J. Cho, unpublished) or pdBhss GN5-6 (M.-J. Cho, unpublished), all containing uidA (ß-glu-curonidase; gus, GUS) under the control of the barley B1- (p16; pdBhGN1-2; pdBhssGN5-6) or D- (pD11-Hor3) hordein promoter and terminated with nos. Cultures were selected with 5 mg L-1 bialaphos on callus-induction medium, either (i) DM medium or (ii) DC medium. Two methods were used to generate the transgenic plants. In the first scheme (Lemaux et al., 1996; Cho et al., 1999), plants from putative transgenic callus lines, obtained after selection of bombarded IEs cultured in the absence of osmotic treatment on DM medium in the dark, were regenerated on solid FHG medium containing 3 mg L-1 bialaphos and exposed to light (approximately 30-50 µmol m-2 s-1). For the second transformation scheme, transgenic calli, produced from bombarded, osmotically treated IEs, were transferred for an intermediate culturing step, between the callus-induction (DC medium) and regeneration (FHG) steps, onto DBC2 medium containing 5 mg L-1 bialaphos (Cho et al., 1998). This intermediate culturing step was carried out under dim light conditions (approximately 10 to 30 µmol m-2 s-1, 16-h light) for 1 to 2 mo. For both schemes regenerated shoots were transferred into Magenta boxes with rooting medium containing 3 mg/L bialaphos. When shoots reached the top of the box, plantlets were transferred to soil in the greenhouse. Transgenic plants were identified by PCR and/or DNA blot hybridization analyses as described (Cho et al., 1998, 1999). Fifty-nine randomly selected independent transgenic lines were used for cytological analysis. Root tips of 1 to 5 plants were examined from each transgenic line.
Cytological Analysis
Random samples of nontransgenic callus tissues were taken at 4, 12, and 20 wk after callus initiation. For cytological analysis, small pieces of callus tissue (2–4 mm), randomly selected and subcultured for 3 to 5 d, were pretreated in saturated 1-bromonaphthalene solution overnight at 4°C, fixed in 1:3 glacial acetic acid:ethanol and stored at 4°C. To observe chromosome numbers, callus tissues were hydrolyzed in 1 M HCl at 60°C for 2 min and stained in Feulgen solution. A squash preparation was made in a drop of 1% (v/v) aceto-carmine. Chromosome numbers from cells in which chromosomes were not well spread or could not be accurately counted were not included in the data set.
For cytological analysis of transgenic or nontransgenic barley plants, healthy root tips were collected from young plants grown in the greenhouse. After pre-treatment in saturated 1-bromonaphthalene solution overnight at 4°C, root meristems were fixed in 1:3 glacial acetic acid:ethanol and stored at 4°C. Root meristems were hydrolyzed in 1 M HCl at 60°C for 5 to 7 min, stained in Feulgen solution and squashed on a glass slide in a drop of 1% aceto-carmine. Chromosomes were counted from at least five well-spread cells per plant.
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Results |
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Discussion |
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TOPABSTRACTINTRODUCTIONMaterials and methodsResultsDiscussionREFERENCES |
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There are several factors that might explain the occasionally observed abnormal ploidy in regenerated, nontransgenic plants. These include factors known to affect chromosomal instability in plant tissues during in vitro growth, such as the particular plant species, genotype, initial ploidy level, explant source, medium composition, growth regulators used, and time in culture (Constantin, 1981; Karp, 1988). For example, the extent of gross chromosomal aberration in barley, i.e., polyploidy and aneuploidy, was shown to be positively correlated with increasing time in culture (Ziauddin and Kasha, 1990; Wang et al., 1992).
In the present study, numerical and structural changes in chromosomes occurred in nontransgenic callus cells at an early stage. Although none of the 149 cells examined in the uncultured IEs had abnormal chromosome numbers, cultured embryos at 1, 3, and 7 d had tetraploid cells at frequencies of 1.7, 3.7, and 2.4%, respectively. At 2 wk in unpassaged callus, in addition to cells with a tetraploid number of chromosomes, cells that were aneuploid or contained structural variations began to increase in number. For transformation, bombardment of the IEs occurred 1 d after culturing began. The percentage of tetraploid cells at that time, 1.7%, is much lower than the percentage of independent transgenic events that were tetraploid, 46%. Therefore, the high percentage of lines giving rise to tetraploid plants is not explained by the pre-existence of large numbers of tetraploid cells in the IEs at the time of bombardment.
At 4 wk, cells from randomly selected callus had extensive chromosomal changes, ranging from haploid (2n = 7) to octaploid (2n = 56); however, the highest percentages of cells were either diploid (68%) or tetraploid (8.6%) with some near-tetraploid. In 3-mo-old, randomly selected nontransgenic callus, comparable in age to that used to generate the nontransgenic and transgenic plants in this study, 23% of the cells were tetraploid. In comparison, only 1.1% of the regenerated, nontransgenic plants were tetraploid.
One possible explanation for this discrepancy in percentage tetraploid might be differences in the selection of tissues that were represented in the two sets of materials. The nontransgenic callus examined in this study derived from tissue that was randomly selected during passage, in contrast to the tissue from which the nontransgenic plants were regenerated, which was selected for its apparent regenerability. Consistent with this explanation, Singh (1986) observed that numerical changes in the chromosomal content occurred in the majority of cells (range = 67–88%) in nonregenerable barley callus cultures, whereas such changes occurred at a much lower frequency (range = 0–26%) in regenerable callus cultures. Thus, the selection of embryogenic cultures for the regeneration of the nontransgenic plants would be expected to give a much lower percentage of cells with chromosomal variation.
The frequency of cytological aberration observed in regenerated, transgenic barley plants in this study was high; 27 of 59 independent transgenic lines (46%) had plants with an altered ploidy level, either tetraploid (2n = 4x = 28) or aneuploid around the tetraploid level (i.e., 26, 27, 29, and 30); no aneuploidy was observed around the diploid number of chromosomes. In hexaploid oat (2n = 6x = 42), a high frequency (60%) of plants with cytogenetic aberrations was also observed in transgenic lines compared with that in nontransgenic oat plants (0–14%; Choi et al., 1999). Both the transgenic and nontransgenic lines were treated in a manner similar to that described for barley in this study except that hygromycin was used as the selection agent for oat transformation. The most common cytogenetic aberration in the transgenic oat plants was aneuploidy, followed by deletion of chromosomal segments; no change in ploidy level was observed. In the case of soybean (2n = 2x = 40), cytological variations, both tetraploidy and aneuploidy around the diploid and tetraploid levels were reported in transgenic soybean plants (Singh et al., 1998). It should be noted, however, that soybean is considered an ancient autotetraploid with much of its genome duplicated and hence "diploid" soybean (2n = 40) may be more tolerant of aneuploidy and ploidy change than a true diploid (Shoemaker et al., 1996). Thus, the particular type of chromosomal aberration, e.g., ploidy level changes versus aneuploidy and the tolerance for aneuploidy around the diploid number of chromosomes, appears to be dependent upon the particular plant species and possibly its inherent ploidy level.
The various stresses that occur during in vitro culturing are known to affect chromosomal stability, e.g., medium composition, plant species and time in culture (Constantin, 1981; Karp, 1988). In our study, the stresses imposed by the transformation process, above those caused by the in vitro-culturing process itself, likely added to or exacerbated the chromosomal instability in tissues from which the transformed plants were regenerated. For example, the DNA introduction process is likely stressful since it involves exposure of cells to vacuum, cellular damage due to microprojectile impact, and potential loss of cell turgor following particle impact. In addition, selection is used to identify transformed tissue (Jähne et al., 1994; Wan and Lemaux, 1994; Funatsuki et al., 1995; Hagio et al., 1995; Salmenkallio-Marttila et al., 1995; Koprek et al., 1996; Lemaux et al., 1996; Tingay et al., 1997; Cho et al., 1998, 1999; Zhang et al., 1999) and, during the selection process, transformed tissue must grow in the presence of dead or dying tissue for prolonged periods, likely causing cellular stress. That the transformation process causes additional impact on the integrity of the chromosome is consistent with the poor field performance observed in replicated field trials of transgenic barley plants (Bregitzer et al. 1998) compared with field trials of regenerated, nontransgenic plants of the same genotype (Bregitzer and Poulson, 1995).
There was no difference in frequency of chromosomal aberration between the two different culturing procedures used for barley transformation. These two schemes included differences in media used for culture initiation, the in vitro culturing scheme used during the selection phase and the use of osmoticum during transformation. One scheme involved the use of DM medium alone for the entire culture initiation and selection periods, with no BAP or elevated copper in the medium. The other scheme involved the use of osmoticum treatment, DC medium for initiation and selection, followed by an intermediate culturing step on BAP- and high copper-containing DBC2 medium. The latter culturing scheme was developed to increase regenerability of cultures. As reported by Dahleen (1995), a high copper-containing initiation medium (DC medium) produced more regenerable tissues than did DM medium. The presence of BAP in the intermediate step-medium leads to the production of more highly regenerable, meristem-like tissues (Cho et al., 1998; Lemaux et al., 1999). The use of this intermediate culturing step was shown to lead to an increase in the regenerability of certain transgenic lines (65%) (unpublished data), compared with the use of DM medium alone without an intermediate culturing step (52–57%) (Wan and Lemaux, 1994; Lemaux et al., 1996). Despite the apparent increase in regenerability with the use of the intermediate step, the frequency of chromosomal abnormalities in the plants obtained by this culturing scheme (45%) was not different from that obtained from culturing on DM medium alone (47%). That a difference is not seen despite the positive effects of the use of the intermediate step on regenerability might indicate that ploidy changes occur early during the selection process prior to the use of the intermediate step.
In the analysis of callus tissue, cells were found that were aneuploid around the diploid number. Among the nontransgenic and transgenic plants, no plants with aneuploid numbers around the diploid number were found; however, relatively high numbers of transgenic plants were aneuploid around the tetraploid level (i.e., 26, 27, 29, and 30). These results are consistent with the results of earlier studies, which showed that in regenerated plants from established diploid and tetraploid cultures of Italian ryegrass, chromosomal aberration was detected only from the tetraploid lines (Jackson and Dale, 1988). In general, plants regenerated from diploid species are more likely to be aneuploid around a polyploid number of chromosomes than a diploid number even though such cells exist in the callus population. This is presumably because the polyploid cells are more buffered to the effects of gene losses in aneuploids because of gene dosage. In the case of the hexaploid species of wheat (2n = 6x = 42), 29% of regenerated plants were aneuploid (2n = 38–45) following in vitro culture of tissue, but no changes in ploidy level were observed (Karp and Maddock, 1984). In hexaploid oat plants (2n = 6x = 42), 15% of regenerated plants from tissues derived from three different media had chromosomal aberration such as aneuploidy and structural changes, but no change in ploidy level was observed (H.W. Choi, P.G. Lemaux, and M.-J. Cho, data not shown).
In addition to chromosomal abnormalities, phenotypic variation was also observed in regenerated transgenic plants and this was related to changes in ploidy level. Tetraploid transgenic plants were different from diploid transgenic plants in growth rate and morphology at both the vegetative and reproductive stages with later heading dates, broader leaves, thicker roots, and longer seeds in tetraploid plants. Similar phenotypic abnormalities, such as larger plant organs in polyploid compared with diploid plants, were reported in regenerated, nontransgenic plants of alfalfa and potato (reviewed by Lee and Phillips, 1988).
In our study, we also observed a higher percentage of tetraploid plants (75%) with low or no seed set compared with the percentage of diploid plants (19%) with low or no seed set. Evans and Rahman (1990) reported similar results with autotetraploid forms of two barley cultivars in which grain yield was less in the polyploid varieties compared with their diploid counterparts due to fewer tillers, fewer florets per spike and lower fertility. Low fertility in tetraploid plants is likely related to instability of chromosome number during an abnormal meiosis. Once established, however, abnormal chromosome numbers can be maintained in tetraploid progeny (T1–T3) and transmitted to subsequent generations; the degree of fertility in general remains the same as in the T0 plants (data not shown).
In the present study, we observed a significantly higher frequency of regenerated, transgenic plants with increased ploidy or gross chromosomal abnormalities (46%) than in regenerated, nontransgenic plants (1.1%). The frequency with which chromosomal abnormalities are seen in the transgenic plants might reflect the generalized trend toward chromosomal abnormality mediated by the increased stresses of the transformation process. The differences are not likely due to the pre-existence of cells with abnormal chromosomes in the embryo since no such cells were found in the uncultured embryo and only 1.7% of the cells were found to be tetraploid at Day 1, when bombardment takes place. That the additional stresses of transformation might lead to increased chromosomal abnormalities is likely since only 23% of cells in randomly selected callus are tetraploid when they are the same age as the tissue used to regenerate transgenic plants, 46% of which are tetraploid or aneuploid.
This tendency toward tetraploidy or aneuploidy in transgenic barley plants could be explained by the fact that, since DNA introduced by bombardment can remain in the cultured cells for at least 2 wk post-bombardment (unpublished data), this situation could lead to an increasing probability of the transgene integrating into cells that have already become tetraploid, especially if the stresses of the transformation process exacerbate ploidy instability. In a previous study (Cho et al., 1999), T0 plants from 6 of 12 independent transgenic barley lines were tetraploid. Out of these six lines, plants from only one line gave a ratio consistent with a 35:1 segregation of GUS expression (driven by the endosperm-specific hordein promoters); plants from the remainder of the lines gave a ratio consistent with a 3:1 segregation of expression. If cells were already tetraploid at the time of DNA integration, it would be expected that transgene segregation would be 3:1; if cells were diploid and became tetraploid after DNA integration, then a 35:1 segregation ratio would be expected.
It is also possible, since DNA is introduced into random cells and since one of the culturing schemes ("intermediate step") is known to increase regenerability, that transformed cells that might otherwise be lost because of cell-division incompetency or lack of regenerability could be "rescued" by this culturing scheme. For example, cells might become tetraploid during the selection process at an increased frequency compared with nonselection conditions and the improved culturing scheme might allow the tetraploid/aneuploid cells to grow competitively and regenerate. That this is not the case is demonstrated by the fact that the percentages of tetraploid events from the two different culturing schemes, including the improved culturing scheme, is approximately the same (47%, DM medium vs. 45%, intermediate step; Table 3).
Although perhaps more exacerbated in barley than other crop species, the increase in chromosomal variation in transgenic plants compared with nontransgenic plants is clearly undesirable when using genetic engineering technologies to effect agronomic improvement. The changes observed in this study in chromosomal number and integrity only quantitate gross changes in chromosomal integrity. It is also likely that other less visible changes in chromosomal fidelity also occur (e.g., mutation, heritable methylation changes) and these also likely impair the ability of the transgenic plants to perform in an identical manner to nontransgenic parental plants. Since barley seems particularly sensitive to the stresses that induce ploidy and other chromosomal aberrations, it becomes an ideal species in which to identify and reduce the stresses that negatively affect both barley and likely other crop species. Identifying and reducing these stresses should lead to the development of methods for generating transgenic plants that are more genetically and agronomically identical to the parent plants, a key goal for the manipulation of crop plants through genetic engineering.
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ACKNOWLEDGMENTS |
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Received for publication February 26, 1999.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMaterials and methodsResultsDiscussionREFERENCES |
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kerman S., Kurtén U., Mannonen L., Ritala A., Teeri T.H., Kauppinen V. Transgenic barley (Hordeum vulgare L.) by electroporation of protoplasts. Plant Cell Rep 1995;15:301-304.This article has been cited by other articles:
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  K. B. R. S. Visarada, K. Meena, C. Aruna, S. Srujana, N. Saikishore, and N. Seetharama Transgenic Breeding: Perspectives and Prospects Crop Sci., August 7, 2009; 49(5): 1555 - 1563. [Abstract] [Full Text] [PDF]
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 J. Li, A. M. Brunner, R. Meilan, and S. H. Strauss Stability of transgenes in trees: expression of two reporter genes in poplar over three field seasons Tree Physiol, February 1, 2009; 29(2): 299 - 312. [Abstract] [Full Text] [PDF]
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P. Bregitzer, L. S. Dahleen, S. Neate, P. Schwarz, and M. Manoharan A Single Backcross Effectively Eliminates Agronomic and Quality Alterations Caused by Somaclonal Variation in Transgenic Barley Crop Sci., March 19, 2008; 48(2): 471 - 479. [Abstract] [Full Text] [PDF]
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P. Bregitzer, A. E. Blechl, D. Fiedler, J. Lin, P. Sebesta, J. F. De Soto, O. Chicaiza, and J. Dubcovsky Changes in High Molecular Weight Glutenin Subunit Composition Can Be Genetically Engineered without Affecting Wheat Agronomic Performance Crop Sci., May 18, 2006; 46(4): 1553 - 1563. [Abstract] [Full Text] [PDF]
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N. B. Leibbrandt and S. J. Snyman Stability of Gene Expression and Agronomic Performance of a Transgenic Herbicide-Resistant Sugarcane Line in South Africa Crop Sci., March 1, 2003; 43(2): 671 - 677. [Abstract] [Full Text] [PDF]
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P. Bregitzer and D. Tonks Inheritance and Expression of Transgenes in Barley Crop Sci., January 1, 2003; 43(1): 4 - 12. [Abstract] [Full Text] [PDF]
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P. Bregitzer, S. Zhang, M.-J. Cho, and P. G. Lemaux Reduced Somaclonal Variation in Barley Is Associated with Culturing Highly Differentiated, Meristematic Tissues Crop Sci., July 1, 2002; 42(4): 1303 - 1308. [Abstract] [Full Text] [PDF]
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