PlantCeU Reports
Plant Cell Reports (1994) 13:652-656
9 Springer-Verlag1994
Easy determination of ploidy level in Arabidopsis thaliana plants by means of pollen size measurement Thomas Altmann 1, Brigitte Damm 1, ,, Wolf B. Frommer 1, Thomas Martin 1, Peter C. Morris 1, Dieter Schweizer 2, Lothar Willmitzer 1, and Renate Schmidt 2' ** 1 Institut ffir Genbiologische Forschung Berlin GmbH, Ihnestrasse 63, 14195 Berlin, Germany 2 Institut for Botanik, Universit/it Wien, Rennweg 14, A-1030 Wien, Austria * Present address: MOGEN International nv, Einsteinweg 97, 2333 CB Leiden, The Netherlands ** Present address: IPSR Cambridge Laboratory, John Innes Centre, Colney Lane, Norwich NR4 7UJ, UK
....
Received 28 June 1993/Revised version received 22 March 1994 - Communicated by H. Lrrz
Summary. Cytogenetic examination of transgenic Arabidopsis thaliana (L.) Heynh. plants obtained by Agrobacterium-mediated gene transfer to cotyledon- and root-explants or by direct gene transfer into protoplasts revealed a high percentage of tetraploid or aneuploid transformants. Depending on the transformation procedure used, 13% (root explant transformation), 33% (cotyledon explant transformation), or 38% (direct gene transfer) of the transformants showed aberrant ploidy levels. A good correlation between the ploidy level of a plant and the size of its pollen grains was observed. This allows quick and simple testing of the ploidy level of transgenic Arabidopsis plants. Key words: Transgenic Arabidopsis - Ploidy - Pollen size
Abbreviations: AM -Arabidopsis medium, ANOVA - analysis of variance, DAPI - 4,6-Diamidino-2phenylindole, PEG - polyethyleneglycol
mutants w i t h altered expression of the reporter gene. Typically in such experiments the promoter of a regulated gene is fused to the coding regions of selectable or screenable reporter genes. Transgenic plants harboring these constructs are mutagenized in order to identify mutations in the signal perception and transduction pathway (Karlin-Neumann et al. 1991; Frommer et al. 1991). As the majority of the expected mutations would lead to a loss of a particular gene function, most of them would appear as recessive mutations. Therefore, a critical prerequisite for the application of such strategies is the use of diploid transgenic plants to enable the isolation of homozygous mutants in the progeny of mutagenized plants. Here we show that a high proportion (13 to 38%) of t r a n s g e n i c A r a b i d o p s i s plants o b t a i n e d by Agrobacterium-mediated or direct gene gene transfer and regeneration through a callus phase are tetraploid or aneuploid. Based on the good correlation between the ploidy of a plant and the size of its pollen grains, we present a quick and easy assay for ploidy level by microscopic analysis of the pollen size.
Introduction Due to its unique properties (reviewed by Meyerowitz 1989) the small weed Arabidopsis thaliana (L.) Heynh. serves as a model system for classical as well as molecular genetics of higher plants. The possibility to stably introduce foreign DNA into the Arabidopsis genome either by Agrobacterium-mediated (Lloyd et al. 1986; Feldmann and Marks 1987; Valvekens et al. 1988; Schmidt and Willmitzer 1988) or by direct gene transfer (Damm et al. 1989; Karesch et al. 1991), made available novel strategies for the isolation of specific mutants. Insertion mutants can for example be obtained by using Agrobacterium-mediated T-DNA transfer (Feldman 1991; Koncz et al. 1989; Van Lijsebettens et al. 1991). I-Ieterologous transposable elements (Schmidt and Willmitzer 1989; Balcells et al. 1991; Altmann et al. 1992) are expected to serve as efficient insertion mutagens. Furthermore, reporter constructs can be introduced that allow the identification or selection of
Correspondence to: T. Altmann
Materials and Methods Plant growth conditions. Arabidopsis thaliana (L.) Heyrth. ecotype C24 (Valvekens et al. 1988) was used in all experiments. Plants for chromosome preparations were grown aseptically on AM medium as described (Schmidt and Willmitzer 1988). Pollen was taken from soil-grown plants, cultured under a 16 h day (3000 lux fluorescent light, 20~ : 8 h night (I7~ regime and a relative humidity of 70%. Transformation of Arabidopsis. Agrobacterium-mediated transformation of leaf or cotyledon explants was performed as described by Schmidt and Willmitzer (1988). Root transformation was done according to Valvekens et al. (1988) with the modification that rooting of regenerated shoots was performed on AM medium (Schrnidt a n d Willmitzer 1988) supplemented with 500 mg/1 Claforan, and 1 gfl activated charcoal. Direct gene transfer and protoplast regeneration was performed as described by Damrn et al.
653 (1989). A line was defined as selfed progeny of a single antibiotic resistant T1 plant derived from a particular primary transformant (T0).
Preparation of metaphase chromosomes. Roots were isolated from young, rapidly growing T2 plants (2 - 3 weeks old) and washed with water. To enrich for metaphases, root tissue was incubated in 2 mM hydroxyquinoline for 2 hours at room temperature and subsequently for 2 hours at 4~ in the dark. The tissue was fixed in ethanol/glacial acetic acid (3:1 v:v) for one hour at room temperature. This step was repeated overnight at 4~ The samples were then rehydrated stepwise (10 min in 50% (v/v) ethanol, 10 rain in 25% (v/v) ethanol, 10 min in water), incubated for 35 min in 5 M HC1 at room temperature and subsequently for 2 hours at room temperature in the dark in Feulgen's solution (Feulgen and Rossenbeck 1924). The roots were washed with water and root tips were isolated in carmin acetic acid (Darlington and laConr 1960) and squashed. The use of both stains was necessary to obtain chromosome preparations with sufficient contrast. Alternatively, roots squashes were prepared by cellulase / pectinase digestion after pretreatment with 2 mM hydroxyquinoline and fixation, and stained with DAPI as described by Maluszynska and Heslop-Harrison (1991). Furthermore, the same protocol was applied to
prepare squashes of immature flower buds (size about 0.5 mm) with the exeption that the cellulase / pectinase digestion was carried out for 1.5 hours. The chromosome preparations were analyzed by light or fluorescence (360370 nm) microscopy (1000-fold magnification). For each line analyzed at least three plants were tested with at least three good chromosome spreads examined per plant.
Determination of pollen size. Single mature flowers of soilgrown plants were tapped a few times onto a slide and pollen grains were immediately (without any storage) measured using a microscope with a scaled ocular (320-fold magnification). For this analysis only well-shaped (ellipsoid) grains were selected, aberrant pollen which more frequently were observed in tetraploid than in diploid plants (data not shown) were omitted. A total of 10 pollen grains (2 x 5) derived from two plants per line were measured.
In vitro pollen germination test. Freshly harvested pollen grains of soil-grown plants were layered onto the surface of standard pollen growth medium as described by Jahnen et al. (1989). After cultivation for 3 to 4 h at 25~ in the dark, the frequency of germinated pollen grains was determined microscopically.
Fig. 1. Metaphase chromosomes stained with Feulgen's reagent in root tip squashes of Arabidopsis seedlings. A) metaphase plate of a diploid plant, B) and B') metaphase plate of a tetraploid plant at two levels of focus. The bars indicate 5 Ixm.
Fig. 2. Pollen grains of A) a diploid plant and B) a tetraploid plant. The bars indicate 50 Ixm.
654 Results
Ploidy of transgenic plants correlates with the size of pollen grains Metaphase chromosomes were prepared from root tips or immature flower buds of plants of independent transgenic Arabidopsis thaliana lines and counted. In plants of diploid lines, 10 chromosomes were observed (Fig. 1 A). Lines showing 20 (or an average of at least 18) chromosomes were termed tetraploid (Fig. 1 B, B'). Due to the small size and high number of chromosomes present in these metaphase plates, it was not always possible to identify 20 individual chromosomes in all ceils examined. A few of the latter lines may therefore be aneuploid rather than truly tetraploid. Cytogenetic examination of 34 randomly chosen lines derived from protoplast transformants identified 20 diploid and 14 tetraploid lines. A further set of 15 independent lines, 8 diploid and 7 tetraploid as tested c y t o g e n e t i c a l l y , obtained by AgrobacteriumTable 1. Sizes of pollen grains of diploid and tetraploid plants diploid a line pollen-length b pollen.width b pollen size e CT3 26.6 + 1.65 15.2 + 1.03 404.3 CT5 28.8 5:1.03 16.2 + 0.63 466.6 CT7 26.8 + 1.40 15.2 + 1.03 407.4 CT8 28.2 + 0.63 15.4 5:0.97 434.3 CT 10 26.8 + 1,69 15.4 + 1.90 412.7 CT 11 26.8 + 1.93 15.8 5:0.63 423.4 CT 12 28.6 + 1.35 14.8 + 1.03 423.3 CT 15 27,8 5:1.84 15.6 5:0.84 430.6 PT 1 27.6 + 1.27 15.8 5:0.63 436.1 PT2 27.4 5:0.97 15.4 5:0.97 422.0 PT4 27.8 + 0.63 15.2 + 1.03 422.6 PT 6 27.2 5:t.40 15.2 + 1.03 413.4 PT7 27.0 + 1.41 14.8 + 1.03 399.6 PT 8 28.0 + 0.00 15.6 + 0.84 436.8 PT9 27.8 5:0.63 15.0 + 1.05 417.0 PT 12 28.0 5:0.00 15.8 5:0.63 442.4 PT 13 27.8 + 0.63 15.8 5:0.63 439.2 PT 16 27.6 _+ 0.84 15.4 5:0.97 425.0 PT 21 28.0 5:0.00 15.4 5:0.97 431.2 PT 22 27.8 5:0.63 15.6 5:0.84 433.7 PT 23 27.6 5:0.84 14.8 + 1.03 408.5 PT 24 28.2 5:0.63 15.4 5:0.97 434.3 PT 25 27.4 5:0.97 14.8 5:1.03 405.5 PT 26 28.2 5:0.63 15.4 5:0.97 434.3 PT 30 27.8 + 0.63 15.2 5:1.03 422.6 PT 31 27.6 • 0.84 16.0 5:0.00 441.6 t ~ 32 27.4 5:0.97 15.6 • 0.84 427.4 PT 33 27.8 5:0.63 15.6 5:0.84 433.7 PT 34 28.0 5:0.00 15.6 + 0.84 436.8
transformation of cotyledons were selected for pollen size determination. The analysis of the 49 lines revealed that pollen of diploid plants was 26.6 - 28.8 Ixm (mean 27.66) in length and 14.8 - 16.2 lxm (mean 15.41) in width, whereas pollen of tetraploid plants was 3 2 . 6 36.6 Ixm (mean 35.30) in length and 18.6 - 21.2 ~tm (mean 19.88) in width (Fig. 2, Table 1). There was a significant difference between the two ploidy levels both for pollen length and width (19 < 0.0001) as determined by A N O V A (performed on the raw data). As a crude measure of pollen size, the product of the mean length and width (longitudinal and latitudinal axes) of the pollen grains was calculated. This value (here defined as pollen size) enabled a clear distinction of diploid from tetraploid plants and thus allowed an easy classification. From the data shown in Table 1 we conclude that a pollen size of < 500 pm 2 is a highly reliable indication for a diploid karyotype and a value of > 600 Ixm2 clearly indicates tetraploidy.
line
tetraploid a pollen-length b pollen.width b
CT 1 CT2 CT4 CT6 CT9 CT 13 CT 14
34.6 34.8 32.6 36.6 34.6 33.6 34.4
+ + + + + + +
1.90 1.93 2.12 1.90 2.12 2.63 2.27
19.6 • 0.84 20.0 + 1.63 21.2 + 2.53 21.0 5:1.70 19.6 + 1.26 20.6 + 2.32 19.2 + 1.69
pollen size c 678.2 696.0 691.1 768.6 678.2 692.2 660,5
PT3 PT5 PT 10 PT 11 PT 14 PT 15 PT 17 PT 18 PT 19 PT 20 PT 27 PT 28 PT 29
35.8 36.0 36.2 35.6 35.8 34.8 36.0 36.0 36.6 35.8 35.6 35.2 35.4
+ 0.63 + 0.00 + 0.63 + 1.27 :k 0.63 + 1.69 + 0.00 + 0.00 5:0.97 5:0.63 + 1.27 + 1.40 5:1.35
20.0 5:0.00 19.8 + 0.63 20.0 5:0.00 19.8 _+ 0.63 20.0 + 0.00 18.6 _+ 0.97 19.8 5:0.63 20.0 + 0.00 19.8 + 0.63 20.0 + 0.00 19.8 5:0.63 19.4 5:0.97 19.4 5:0.97
716.0 712.8 724.0 704.9 716.0 647.3 712.8 720.0 724.7 716.0 704.9 682.9 686.8
The sizes of pollen grains of stably transformed and cytologically tested plants were determined microscopically. Lines obtained by cotyledon transformation were designated with the prefix CT, lines derived from l~rotoplast _transformants were labeled with the prefix PT. a To establish the ploidy level of the lines plants were examined cytologically. b Maximal length and width (in p.m) of 10 pollen grains tested for each line were measured. The mean values and the standard deviations are given. c As a crude measure, the pollen size was calculated as the product of the means of longitudinal and latitudinal axes of the pollen grains (I.tm2).
655 Table 2. Determination of the ploidy level from the pollen size (PS) line CT16 CT17 CT 18 CT19 CT20 CT21 CT22 CT23 CT24 CT25 CT26 CT27 CT28 CT29 CT30 CT31 CT32 CT33
PS ploidy 412.8 2n 708.8 4n 472.0 2n 431.2 2n 705.6 4n 443.0 2n 443.2 2n 412.8 2n 425.6 2n 689.6 4n 465.6 2n 430.4 2n 784.0 4n 430.4 2n 435.2 2n 721.3 4n 476.8 2n 390.4 2n
line CT34 CT35 CT36 CT37 CT38 CT39 CT40 CT41 CT42 CT43 CT44 CT45 CT46 CT47 CT48 CT49 CT50 CT51
PS ploidy 712.0 4n 369.6 2n 744.0 4n 448.0 2n 390.4 2n 684.5 4n 685.4 4n 483.2 2n 448.0 2n 436.8 2n 392.0 2n 478.9 2n 457.6 2n 720.0 4n 440.0 2n 403.2 2n 425.6 2n 425.6 2n
line CT52 CT53 CT54 CT55 CT56 CT57 CT58 CT59 CT60 CT61 CT62 CT63 CT64 CT65 CT66 CT67
PS ploidy 720.0 4n] 697.8 4n 492.8 2n 472.0 2n 448.0 2n 448.0 2n 446.1 2n 720.0 4n 347.2 2n 750.7 4n 775.2 4n 425.6 2n 472.3 2n 364.0 2n 728.0 4n 705.6 4n
line RT1 RT2 RT3 RT4 RT5 RT6 RT7 RT8 RT9 RT 10 RTI1 RT12 RT13 RT14 RT15 RT16 RT17 RT 18
PS ploidy 355.9 2n 417,2 2n 508.7 2n 445.3 2n 402.3 2n 366.5 2n 666.3 4n 423.2 2n 416.1 2n 437.3 2n 419.1 2n 392.2 2n 479.7 2n 398.8 2n 730.6 4n 793.0 4n 421.4 2n 404.3 2n
line RT19 RT20 RT21 RT22 RT23 RT24 RT25 RT26 RT27 RT28 RT29 RT30 RT31 RT32 RT33 RT34 RT35 RT36
PS ploidy 427.6 2n 439.6 2n 437.9 2n 412.7 2n 456.5 2n 768.2 4n 447.9 2n 512.4 2n 464.8 2n 429.3 2n 401.7 2n 417.2 2n 467.1 2n 484.4 2n 459.5 2n 417.2 2n 523.3 2n 467.5 2n
line RT37 RT38 RT39 RT40 RT41 RT42 RT43
PS ploidy 387.4 2n 725.0 4n 770.9 4n 392.0 2n 431.2 2n 497.9 2n 431.7 2n
Lines with the label CT are cotyledon transformants, those with the prefix RT are root transformants. The sizes of 10 pollen grains from each line were determined microscopically and used to deduce the ploidy levels (see results section and Table 1). The pollen size of three lines (RT 3, RT 26, RT 35) slightly exceeds the maximal size of 500 gm 2 previously determined for pollen of diploid plants. These lines were nevertheless classified as diploid as their pollen sizes were clearly below those of tetraploid plants (> 600 Ixm2) andin one case ploidy level was confirmed independently by chromosome counting. The viability of the pollen grains was examined by germination test of pollen isolated from plants of 11 diploid lines and 10 tetraploid lines and untransformed control (C24). Germination frequencies varied between different transformed lines and in general were lower for pollen derived from tetraploid plants: C24 showed 74% germinated pollen grains, for the diploid lines 55%, 76%, 78%, 80%, 77%, 60%, 77%, 70%, 75%, 66%, and 74% germination, respectively, was observed which is similar to (or only slightly lower than) data reported for other Arabidopsis thaliana races (Pickett 1988; Azarov et al. 1990). The reduced pollen germination frequency of 65%, 79%, 50%, 52%, 45%, 49%, 60%, 41%, 30%, and 52% observed for tetraploid plants, respectively, probably reflects the presence of an increased fraction of aberrant pollen grains (data not shown).
plants. These lines were nevertheless classified as 2n as their pollen sizes were far below those of tetraploid plants (< 600 I.tm2). F u r t h e r m o r e , cytogenetic examination of one of these plants showed the presence of 10 chromosomes. From a total of 34 transgenic lines obtained by protoplast transformation 13 (38%) were tetraploid. Thus, all three transformation procedures yielded tetraploid transgenic plants. The relatively small fraction of 13% tetraploid transformants generated by root transformation compared to 33% and 38% of tetraploid cotyledon or protoplast transformants, respectively, may indicate that different transformation procedures affect the frequency of tetraploid plants.
Frequency of tetraploid plants generated by different transformationprocedures
Transgenic Arabidopsis thaliana plants obtained by three different transformation procedures, Agrobacteriummediated DNA transfer to cotyledon and root explants as well as PEG-mediated direct DNA transfer to protoplasts were tested for their ploidy levels. By cytological analysis, diploid and tetraploid transformants were identified among cotyledon and protoplast transformants. These plants were then tested for pollen size. In agreement with earlier reports (Bronckers 1963; Bouharmont 1965; Balkema 1971), a significant difference for the size of their pollen was observed between plants of the two ploidy levels. The pollen size analysis revealed that only 60 to 70% of the transformants obtained by cotyledon transformation and by direct DNA transfer to leaf mesophyll protoplasts retained the diploid caryotype of the Arabidopsis ecotype C24 used as a source of explants and protoplasts. In
To determine the fraction of tetraploid transformants resulting from different transformation procedures, transgenic Arabidopsis lines derived from Agrobacteriummediated transformations of cotyledon and root explants were analyzed for pollen size (Table 2) in addition to those generated by protoplast transformation (Table 1). O f 52 independent transgenic lines obtained by transformation of cotyledon explants, 35 were diploid and 17 (33%) were tetraploid. In contrast, 39 of 45 independent lines derived from transformation of root explants by Agrobacterium appeared diploid, the other 6 lines (13%) were tetraploid. Three lines were observed in which the pollen size slightly exceeded the value of 500 ~tm 2 previously determined as diagnostic of diploid
Discussion
656 contrast, 87% of the transformants derived from root transformation were diploid. Tissue culture steps involved in all three transformation procedures have been reported to induce gross karytope changes as well as chromosome rearrangements in a variety of plant species (Larkin and Scowcroft 1981; Karp and Bright 1985; Lee and Philips 1988). In contrast, no change in ploidy level was observed in 120 transformants obtained by embryo transformation of Arabidopsis thaliana (Sangwan et al. 1991). The data presented here do not allow a conclusion as to whether a change in ploidy was induced during the phase of proliferation and regeneration from primarily diploid cells or whether a subset of cells in the explants already contained an increased ploidy level prior to the start of the tissue culture as has been shown for tomato (van den Bulk et al. 1990). Both explanations are possible, as leaf tissue of Arabidopsis has been shown to comprise mixtures of cells with different ploidy levels (Galbraith et al. 1991). The fraction of tetraploid transformants increased with the length of time from the onset of the callus phase until shoot formation (unpublished results). This may reflect a slower regeneration of tetraploid cells than their diploid counterparts in the primary explant or an induction of polyploidy during the callus phase. Nevertheless, t h e comparatively high proportion of tetraploid (or aneuploid) transformants has strong implications for the use of transgenic Arabidopsis plants for mutagenesis, as only diploid lines will be readily useful. These results emphasize the need to test transformants for their ploidy level prior to further use in genetic studies. Due to their small size, the preparation and counting of metaphase chromosomes is tedious and time consuming, especially if many transformants have to be screened. The results shown here allow the use of a quick and easy test for the ploidy level based on the measurement of the size of a few pollen grains per plant. This test is also simpler than counting the number o f guard cell chloroplasts, that has been shown to correlate with the ploidy level of transgenic tomato plants (Jacobs and Yoder 1989). However, minor aneuploidies such as trisomy which in turn causes distinct morphological characteristics in Arabidopsis (Koornneef and Van der Veen 1983) may not be identifed by the pollen size test. Morphological differences between plants of the two ploidy levels examined in the study presented here were observed (but not quantified) in the size of leaves and flowers as well as total height of the plants being larger in case of tetraploid plants. Furthermore, tetraploid plants started flowering two to three weeks later than diploid plants with an accordingly increased number of rosette leaves (data not shown).
Acknowledgements. We would like to thank Sabine Hummel and Carola Recknagel for expert technical assistance. This work was supported by grants from the Bundesministerium f~ir Forschung und Technologie, FRG.
References Altmarm T, Schrnidt R, Willmitzer L (1992) Theor Appl Genet 84:371-383 Azarov AS, Tokarey BI, Netchepurenko AE (1990) Arab Inf Serv 27:9-12 Balcells L, Swinburne J, Coupland G (1991) Tibtech 9: 3136 Balkema GH (1971) PhD Thesis, Wageningen Agricultural University Bouharmont J (1965) In G. R6bbelen (ed), Arabidopsis Research (Symp. G6ttingen), pp 31-36 Bronckers F (1963) Pollen Spores 5:233-238 Datum B, Schmidt R, Willmitzer L (1989) Mol Gen Genet 217:6-12 Darlington CD, laCour LF (1960) The Handling of Chromosomes, Ed. 3, Macmillan, New York Feldman KA (1991) Plant J 1:71-82 Feldmarm KA, Marks MD (1987) Mol Gen Genet 208:1-9 Feulgen R, Rossenbeck H (1924) Z Physiol Chem 135: 203248 Frommer WB, Martin T, Schmidt R, Hummel S, Willmitzer L (1991) In: Bonnemain JL, Delrot S, Lucas WJ, Dainty J (eds) Recent advances in phloem transport and assimilate compartmentation, Ouest Editions, Nantes, pp 254-257 Galbraith DW, Harkins KR, Knapp S (1991) Plant Physiol 96:985-989 Jacobs JP, Yoder JI (1989) Plant Cell Rep 7:662-664 Jahnen W, Lush WM, Clarke AE (1989) Plant Cell 1: 501510 Karesch H, Bilang R, Mittelsten-Scheid O, Potrykus I (1991) Plant Cell Rep 9:571-574 Karlin-Neumann GA, Brusslan JA, Tobin EM (1991) Plant Cell 3:573-582 Karl3 A, Bright SWJ (1985) In: Miflin BJ (ed) Oxford surveys of plant molecular and cell biology, vol. 2. Oxford University Press, Oxford, pp 199-234 Koncz C, Martini N, Mayerhofer R, Koncz-Kalman Z, KOrber H, R6dei GP, Schell J (1989) Proc Natl Acad Sci USA 86:8467-8471 Koornneef M, Van der Veen JH (1983) Genetica 61:41-46 Larkin PJ, Scowcroft WR (1981) Theor Appl Genet 60: 197214 Lee M, Phillips RL (1988) Ann Rev Plant Physiol Plant Mol Biol 39:413-437 Lloyd AM, Barnason AR, Rogers SG, Byrne MC, Fraley RT, Horsch RB (1986) Science 234:464-466 Maluszynska J, Heslop-Harrison JS (1991) Plant J. 1: 159166 Meyerowitz EM (1989) Cell 56:263-269 Pickert M (1988) Arab Inf Serv 26:39-42 Sangwan RS, Bourgeois Y, Sangwan-Norreel BS (1991) Mol Gen Genet 230:475-485 Schmidt R, Willmitzer L (1988) Plant Cell Rep 7:583-586 Schmidt R, Willmitzer L (1989) Mol Gen Genet 220:17-24 Valvekens D, Van Montagu M, Van Lijsebettens M (1988) Proc Natl .&cad Sci USA 85:5536-5540 van den Bulk RW, l.,6ffler HJM, Lindhout WH, Koornneef M (1990) Theor Appl Genet 80:817-825 Van Lijsebettens M, Den Boer B, Hernalsteens J-P, Van Montagu M (1991) Plant Science 80:27-37
Plant Cell Reports (1994) 13:652-656
9 Springer-Verlag1994
Easy determination of ploidy level in Arabidopsis thaliana plants by means of pollen size measurement Thomas Altmann 1, Brigitte Damm 1, ,, Wolf B. Frommer 1, Thomas Martin 1, Peter C. Morris 1, Dieter Schweizer 2, Lothar Willmitzer 1, and Renate Schmidt 2' ** 1 Institut ffir Genbiologische Forschung Berlin GmbH, Ihnestrasse 63, 14195 Berlin, Germany 2 Institut for Botanik, Universit/it Wien, Rennweg 14, A-1030 Wien, Austria * Present address: MOGEN International nv, Einsteinweg 97, 2333 CB Leiden, The Netherlands ** Present address: IPSR Cambridge Laboratory, John Innes Centre, Colney Lane, Norwich NR4 7UJ, UK
....
Received 28 June 1993/Revised version received 22 March 1994 - Communicated by H. Lrrz
Summary. Cytogenetic examination of transgenic Arabidopsis thaliana (L.) Heynh. plants obtained by Agrobacterium-mediated gene transfer to cotyledon- and root-explants or by direct gene transfer into protoplasts revealed a high percentage of tetraploid or aneuploid transformants. Depending on the transformation procedure used, 13% (root explant transformation), 33% (cotyledon explant transformation), or 38% (direct gene transfer) of the transformants showed aberrant ploidy levels. A good correlation between the ploidy level of a plant and the size of its pollen grains was observed. This allows quick and simple testing of the ploidy level of transgenic Arabidopsis plants. Key words: Transgenic Arabidopsis - Ploidy - Pollen size
Abbreviations: AM -Arabidopsis medium, ANOVA - analysis of variance, DAPI - 4,6-Diamidino-2phenylindole, PEG - polyethyleneglycol
mutants w i t h altered expression of the reporter gene. Typically in such experiments the promoter of a regulated gene is fused to the coding regions of selectable or screenable reporter genes. Transgenic plants harboring these constructs are mutagenized in order to identify mutations in the signal perception and transduction pathway (Karlin-Neumann et al. 1991; Frommer et al. 1991). As the majority of the expected mutations would lead to a loss of a particular gene function, most of them would appear as recessive mutations. Therefore, a critical prerequisite for the application of such strategies is the use of diploid transgenic plants to enable the isolation of homozygous mutants in the progeny of mutagenized plants. Here we show that a high proportion (13 to 38%) of t r a n s g e n i c A r a b i d o p s i s plants o b t a i n e d by Agrobacterium-mediated or direct gene gene transfer and regeneration through a callus phase are tetraploid or aneuploid. Based on the good correlation between the ploidy of a plant and the size of its pollen grains, we present a quick and easy assay for ploidy level by microscopic analysis of the pollen size.
Introduction Due to its unique properties (reviewed by Meyerowitz 1989) the small weed Arabidopsis thaliana (L.) Heynh. serves as a model system for classical as well as molecular genetics of higher plants. The possibility to stably introduce foreign DNA into the Arabidopsis genome either by Agrobacterium-mediated (Lloyd et al. 1986; Feldmann and Marks 1987; Valvekens et al. 1988; Schmidt and Willmitzer 1988) or by direct gene transfer (Damm et al. 1989; Karesch et al. 1991), made available novel strategies for the isolation of specific mutants. Insertion mutants can for example be obtained by using Agrobacterium-mediated T-DNA transfer (Feldman 1991; Koncz et al. 1989; Van Lijsebettens et al. 1991). I-Ieterologous transposable elements (Schmidt and Willmitzer 1989; Balcells et al. 1991; Altmann et al. 1992) are expected to serve as efficient insertion mutagens. Furthermore, reporter constructs can be introduced that allow the identification or selection of
Correspondence to: T. Altmann
Materials and Methods Plant growth conditions. Arabidopsis thaliana (L.) Heyrth. ecotype C24 (Valvekens et al. 1988) was used in all experiments. Plants for chromosome preparations were grown aseptically on AM medium as described (Schmidt and Willmitzer 1988). Pollen was taken from soil-grown plants, cultured under a 16 h day (3000 lux fluorescent light, 20~ : 8 h night (I7~ regime and a relative humidity of 70%. Transformation of Arabidopsis. Agrobacterium-mediated transformation of leaf or cotyledon explants was performed as described by Schmidt and Willmitzer (1988). Root transformation was done according to Valvekens et al. (1988) with the modification that rooting of regenerated shoots was performed on AM medium (Schrnidt a n d Willmitzer 1988) supplemented with 500 mg/1 Claforan, and 1 gfl activated charcoal. Direct gene transfer and protoplast regeneration was performed as described by Damrn et al.
653 (1989). A line was defined as selfed progeny of a single antibiotic resistant T1 plant derived from a particular primary transformant (T0).
Preparation of metaphase chromosomes. Roots were isolated from young, rapidly growing T2 plants (2 - 3 weeks old) and washed with water. To enrich for metaphases, root tissue was incubated in 2 mM hydroxyquinoline for 2 hours at room temperature and subsequently for 2 hours at 4~ in the dark. The tissue was fixed in ethanol/glacial acetic acid (3:1 v:v) for one hour at room temperature. This step was repeated overnight at 4~ The samples were then rehydrated stepwise (10 min in 50% (v/v) ethanol, 10 rain in 25% (v/v) ethanol, 10 min in water), incubated for 35 min in 5 M HC1 at room temperature and subsequently for 2 hours at room temperature in the dark in Feulgen's solution (Feulgen and Rossenbeck 1924). The roots were washed with water and root tips were isolated in carmin acetic acid (Darlington and laConr 1960) and squashed. The use of both stains was necessary to obtain chromosome preparations with sufficient contrast. Alternatively, roots squashes were prepared by cellulase / pectinase digestion after pretreatment with 2 mM hydroxyquinoline and fixation, and stained with DAPI as described by Maluszynska and Heslop-Harrison (1991). Furthermore, the same protocol was applied to
prepare squashes of immature flower buds (size about 0.5 mm) with the exeption that the cellulase / pectinase digestion was carried out for 1.5 hours. The chromosome preparations were analyzed by light or fluorescence (360370 nm) microscopy (1000-fold magnification). For each line analyzed at least three plants were tested with at least three good chromosome spreads examined per plant.
Determination of pollen size. Single mature flowers of soilgrown plants were tapped a few times onto a slide and pollen grains were immediately (without any storage) measured using a microscope with a scaled ocular (320-fold magnification). For this analysis only well-shaped (ellipsoid) grains were selected, aberrant pollen which more frequently were observed in tetraploid than in diploid plants (data not shown) were omitted. A total of 10 pollen grains (2 x 5) derived from two plants per line were measured.
In vitro pollen germination test. Freshly harvested pollen grains of soil-grown plants were layered onto the surface of standard pollen growth medium as described by Jahnen et al. (1989). After cultivation for 3 to 4 h at 25~ in the dark, the frequency of germinated pollen grains was determined microscopically.
Fig. 1. Metaphase chromosomes stained with Feulgen's reagent in root tip squashes of Arabidopsis seedlings. A) metaphase plate of a diploid plant, B) and B') metaphase plate of a tetraploid plant at two levels of focus. The bars indicate 5 Ixm.
Fig. 2. Pollen grains of A) a diploid plant and B) a tetraploid plant. The bars indicate 50 Ixm.
654 Results
Ploidy of transgenic plants correlates with the size of pollen grains Metaphase chromosomes were prepared from root tips or immature flower buds of plants of independent transgenic Arabidopsis thaliana lines and counted. In plants of diploid lines, 10 chromosomes were observed (Fig. 1 A). Lines showing 20 (or an average of at least 18) chromosomes were termed tetraploid (Fig. 1 B, B'). Due to the small size and high number of chromosomes present in these metaphase plates, it was not always possible to identify 20 individual chromosomes in all ceils examined. A few of the latter lines may therefore be aneuploid rather than truly tetraploid. Cytogenetic examination of 34 randomly chosen lines derived from protoplast transformants identified 20 diploid and 14 tetraploid lines. A further set of 15 independent lines, 8 diploid and 7 tetraploid as tested c y t o g e n e t i c a l l y , obtained by AgrobacteriumTable 1. Sizes of pollen grains of diploid and tetraploid plants diploid a line pollen-length b pollen.width b pollen size e CT3 26.6 + 1.65 15.2 + 1.03 404.3 CT5 28.8 5:1.03 16.2 + 0.63 466.6 CT7 26.8 + 1.40 15.2 + 1.03 407.4 CT8 28.2 + 0.63 15.4 5:0.97 434.3 CT 10 26.8 + 1,69 15.4 + 1.90 412.7 CT 11 26.8 + 1.93 15.8 5:0.63 423.4 CT 12 28.6 + 1.35 14.8 + 1.03 423.3 CT 15 27,8 5:1.84 15.6 5:0.84 430.6 PT 1 27.6 + 1.27 15.8 5:0.63 436.1 PT2 27.4 5:0.97 15.4 5:0.97 422.0 PT4 27.8 + 0.63 15.2 + 1.03 422.6 PT 6 27.2 5:t.40 15.2 + 1.03 413.4 PT7 27.0 + 1.41 14.8 + 1.03 399.6 PT 8 28.0 + 0.00 15.6 + 0.84 436.8 PT9 27.8 5:0.63 15.0 + 1.05 417.0 PT 12 28.0 5:0.00 15.8 5:0.63 442.4 PT 13 27.8 + 0.63 15.8 5:0.63 439.2 PT 16 27.6 _+ 0.84 15.4 5:0.97 425.0 PT 21 28.0 5:0.00 15.4 5:0.97 431.2 PT 22 27.8 5:0.63 15.6 5:0.84 433.7 PT 23 27.6 5:0.84 14.8 + 1.03 408.5 PT 24 28.2 5:0.63 15.4 5:0.97 434.3 PT 25 27.4 5:0.97 14.8 5:1.03 405.5 PT 26 28.2 5:0.63 15.4 5:0.97 434.3 PT 30 27.8 + 0.63 15.2 5:1.03 422.6 PT 31 27.6 • 0.84 16.0 5:0.00 441.6 t ~ 32 27.4 5:0.97 15.6 • 0.84 427.4 PT 33 27.8 5:0.63 15.6 5:0.84 433.7 PT 34 28.0 5:0.00 15.6 + 0.84 436.8
transformation of cotyledons were selected for pollen size determination. The analysis of the 49 lines revealed that pollen of diploid plants was 26.6 - 28.8 Ixm (mean 27.66) in length and 14.8 - 16.2 lxm (mean 15.41) in width, whereas pollen of tetraploid plants was 3 2 . 6 36.6 Ixm (mean 35.30) in length and 18.6 - 21.2 ~tm (mean 19.88) in width (Fig. 2, Table 1). There was a significant difference between the two ploidy levels both for pollen length and width (19 < 0.0001) as determined by A N O V A (performed on the raw data). As a crude measure of pollen size, the product of the mean length and width (longitudinal and latitudinal axes) of the pollen grains was calculated. This value (here defined as pollen size) enabled a clear distinction of diploid from tetraploid plants and thus allowed an easy classification. From the data shown in Table 1 we conclude that a pollen size of < 500 pm 2 is a highly reliable indication for a diploid karyotype and a value of > 600 Ixm2 clearly indicates tetraploidy.
line
tetraploid a pollen-length b pollen.width b
CT 1 CT2 CT4 CT6 CT9 CT 13 CT 14
34.6 34.8 32.6 36.6 34.6 33.6 34.4
+ + + + + + +
1.90 1.93 2.12 1.90 2.12 2.63 2.27
19.6 • 0.84 20.0 + 1.63 21.2 + 2.53 21.0 5:1.70 19.6 + 1.26 20.6 + 2.32 19.2 + 1.69
pollen size c 678.2 696.0 691.1 768.6 678.2 692.2 660,5
PT3 PT5 PT 10 PT 11 PT 14 PT 15 PT 17 PT 18 PT 19 PT 20 PT 27 PT 28 PT 29
35.8 36.0 36.2 35.6 35.8 34.8 36.0 36.0 36.6 35.8 35.6 35.2 35.4
+ 0.63 + 0.00 + 0.63 + 1.27 :k 0.63 + 1.69 + 0.00 + 0.00 5:0.97 5:0.63 + 1.27 + 1.40 5:1.35
20.0 5:0.00 19.8 + 0.63 20.0 5:0.00 19.8 _+ 0.63 20.0 + 0.00 18.6 _+ 0.97 19.8 5:0.63 20.0 + 0.00 19.8 + 0.63 20.0 + 0.00 19.8 5:0.63 19.4 5:0.97 19.4 5:0.97
716.0 712.8 724.0 704.9 716.0 647.3 712.8 720.0 724.7 716.0 704.9 682.9 686.8
The sizes of pollen grains of stably transformed and cytologically tested plants were determined microscopically. Lines obtained by cotyledon transformation were designated with the prefix CT, lines derived from l~rotoplast _transformants were labeled with the prefix PT. a To establish the ploidy level of the lines plants were examined cytologically. b Maximal length and width (in p.m) of 10 pollen grains tested for each line were measured. The mean values and the standard deviations are given. c As a crude measure, the pollen size was calculated as the product of the means of longitudinal and latitudinal axes of the pollen grains (I.tm2).
655 Table 2. Determination of the ploidy level from the pollen size (PS) line CT16 CT17 CT 18 CT19 CT20 CT21 CT22 CT23 CT24 CT25 CT26 CT27 CT28 CT29 CT30 CT31 CT32 CT33
PS ploidy 412.8 2n 708.8 4n 472.0 2n 431.2 2n 705.6 4n 443.0 2n 443.2 2n 412.8 2n 425.6 2n 689.6 4n 465.6 2n 430.4 2n 784.0 4n 430.4 2n 435.2 2n 721.3 4n 476.8 2n 390.4 2n
line CT34 CT35 CT36 CT37 CT38 CT39 CT40 CT41 CT42 CT43 CT44 CT45 CT46 CT47 CT48 CT49 CT50 CT51
PS ploidy 712.0 4n 369.6 2n 744.0 4n 448.0 2n 390.4 2n 684.5 4n 685.4 4n 483.2 2n 448.0 2n 436.8 2n 392.0 2n 478.9 2n 457.6 2n 720.0 4n 440.0 2n 403.2 2n 425.6 2n 425.6 2n
line CT52 CT53 CT54 CT55 CT56 CT57 CT58 CT59 CT60 CT61 CT62 CT63 CT64 CT65 CT66 CT67
PS ploidy 720.0 4n] 697.8 4n 492.8 2n 472.0 2n 448.0 2n 448.0 2n 446.1 2n 720.0 4n 347.2 2n 750.7 4n 775.2 4n 425.6 2n 472.3 2n 364.0 2n 728.0 4n 705.6 4n
line RT1 RT2 RT3 RT4 RT5 RT6 RT7 RT8 RT9 RT 10 RTI1 RT12 RT13 RT14 RT15 RT16 RT17 RT 18
PS ploidy 355.9 2n 417,2 2n 508.7 2n 445.3 2n 402.3 2n 366.5 2n 666.3 4n 423.2 2n 416.1 2n 437.3 2n 419.1 2n 392.2 2n 479.7 2n 398.8 2n 730.6 4n 793.0 4n 421.4 2n 404.3 2n
line RT19 RT20 RT21 RT22 RT23 RT24 RT25 RT26 RT27 RT28 RT29 RT30 RT31 RT32 RT33 RT34 RT35 RT36
PS ploidy 427.6 2n 439.6 2n 437.9 2n 412.7 2n 456.5 2n 768.2 4n 447.9 2n 512.4 2n 464.8 2n 429.3 2n 401.7 2n 417.2 2n 467.1 2n 484.4 2n 459.5 2n 417.2 2n 523.3 2n 467.5 2n
line RT37 RT38 RT39 RT40 RT41 RT42 RT43
PS ploidy 387.4 2n 725.0 4n 770.9 4n 392.0 2n 431.2 2n 497.9 2n 431.7 2n
Lines with the label CT are cotyledon transformants, those with the prefix RT are root transformants. The sizes of 10 pollen grains from each line were determined microscopically and used to deduce the ploidy levels (see results section and Table 1). The pollen size of three lines (RT 3, RT 26, RT 35) slightly exceeds the maximal size of 500 gm 2 previously determined for pollen of diploid plants. These lines were nevertheless classified as diploid as their pollen sizes were clearly below those of tetraploid plants (> 600 Ixm2) andin one case ploidy level was confirmed independently by chromosome counting. The viability of the pollen grains was examined by germination test of pollen isolated from plants of 11 diploid lines and 10 tetraploid lines and untransformed control (C24). Germination frequencies varied between different transformed lines and in general were lower for pollen derived from tetraploid plants: C24 showed 74% germinated pollen grains, for the diploid lines 55%, 76%, 78%, 80%, 77%, 60%, 77%, 70%, 75%, 66%, and 74% germination, respectively, was observed which is similar to (or only slightly lower than) data reported for other Arabidopsis thaliana races (Pickett 1988; Azarov et al. 1990). The reduced pollen germination frequency of 65%, 79%, 50%, 52%, 45%, 49%, 60%, 41%, 30%, and 52% observed for tetraploid plants, respectively, probably reflects the presence of an increased fraction of aberrant pollen grains (data not shown).
plants. These lines were nevertheless classified as 2n as their pollen sizes were far below those of tetraploid plants (< 600 I.tm2). F u r t h e r m o r e , cytogenetic examination of one of these plants showed the presence of 10 chromosomes. From a total of 34 transgenic lines obtained by protoplast transformation 13 (38%) were tetraploid. Thus, all three transformation procedures yielded tetraploid transgenic plants. The relatively small fraction of 13% tetraploid transformants generated by root transformation compared to 33% and 38% of tetraploid cotyledon or protoplast transformants, respectively, may indicate that different transformation procedures affect the frequency of tetraploid plants.
Frequency of tetraploid plants generated by different transformationprocedures
Transgenic Arabidopsis thaliana plants obtained by three different transformation procedures, Agrobacteriummediated DNA transfer to cotyledon and root explants as well as PEG-mediated direct DNA transfer to protoplasts were tested for their ploidy levels. By cytological analysis, diploid and tetraploid transformants were identified among cotyledon and protoplast transformants. These plants were then tested for pollen size. In agreement with earlier reports (Bronckers 1963; Bouharmont 1965; Balkema 1971), a significant difference for the size of their pollen was observed between plants of the two ploidy levels. The pollen size analysis revealed that only 60 to 70% of the transformants obtained by cotyledon transformation and by direct DNA transfer to leaf mesophyll protoplasts retained the diploid caryotype of the Arabidopsis ecotype C24 used as a source of explants and protoplasts. In
To determine the fraction of tetraploid transformants resulting from different transformation procedures, transgenic Arabidopsis lines derived from Agrobacteriummediated transformations of cotyledon and root explants were analyzed for pollen size (Table 2) in addition to those generated by protoplast transformation (Table 1). O f 52 independent transgenic lines obtained by transformation of cotyledon explants, 35 were diploid and 17 (33%) were tetraploid. In contrast, 39 of 45 independent lines derived from transformation of root explants by Agrobacterium appeared diploid, the other 6 lines (13%) were tetraploid. Three lines were observed in which the pollen size slightly exceeded the value of 500 ~tm 2 previously determined as diagnostic of diploid
Discussion
656 contrast, 87% of the transformants derived from root transformation were diploid. Tissue culture steps involved in all three transformation procedures have been reported to induce gross karytope changes as well as chromosome rearrangements in a variety of plant species (Larkin and Scowcroft 1981; Karp and Bright 1985; Lee and Philips 1988). In contrast, no change in ploidy level was observed in 120 transformants obtained by embryo transformation of Arabidopsis thaliana (Sangwan et al. 1991). The data presented here do not allow a conclusion as to whether a change in ploidy was induced during the phase of proliferation and regeneration from primarily diploid cells or whether a subset of cells in the explants already contained an increased ploidy level prior to the start of the tissue culture as has been shown for tomato (van den Bulk et al. 1990). Both explanations are possible, as leaf tissue of Arabidopsis has been shown to comprise mixtures of cells with different ploidy levels (Galbraith et al. 1991). The fraction of tetraploid transformants increased with the length of time from the onset of the callus phase until shoot formation (unpublished results). This may reflect a slower regeneration of tetraploid cells than their diploid counterparts in the primary explant or an induction of polyploidy during the callus phase. Nevertheless, t h e comparatively high proportion of tetraploid (or aneuploid) transformants has strong implications for the use of transgenic Arabidopsis plants for mutagenesis, as only diploid lines will be readily useful. These results emphasize the need to test transformants for their ploidy level prior to further use in genetic studies. Due to their small size, the preparation and counting of metaphase chromosomes is tedious and time consuming, especially if many transformants have to be screened. The results shown here allow the use of a quick and easy test for the ploidy level based on the measurement of the size of a few pollen grains per plant. This test is also simpler than counting the number o f guard cell chloroplasts, that has been shown to correlate with the ploidy level of transgenic tomato plants (Jacobs and Yoder 1989). However, minor aneuploidies such as trisomy which in turn causes distinct morphological characteristics in Arabidopsis (Koornneef and Van der Veen 1983) may not be identifed by the pollen size test. Morphological differences between plants of the two ploidy levels examined in the study presented here were observed (but not quantified) in the size of leaves and flowers as well as total height of the plants being larger in case of tetraploid plants. Furthermore, tetraploid plants started flowering two to three weeks later than diploid plants with an accordingly increased number of rosette leaves (data not shown).
Acknowledgements. We would like to thank Sabine Hummel and Carola Recknagel for expert technical assistance. This work was supported by grants from the Bundesministerium f~ir Forschung und Technologie, FRG.
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