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Evidence for a constancy of the DNA synthetic period between diploid-polyploid groups in plants
0 1968
by i\catiemic
Experimental
Press Inc.
Cell Research
53, 145-154
(1968)
l-15
EVIDENCE FOR A CONSTANCY OF THE DNA SYNTHETIC PERIOD BETWEEN DIPLOID-POLYPLOID GROUPS IN PLANTS1 MARIA Biology
Department,
R. TROY2 University Received
and
D. E. WIMBER
of Oregon, March
Eugene,
Oreg.
97403,
USA
18, 1968
THE relative durations of the subdivisions of the nuclear cycle have been studied intensively in recent years in a large number of organisms. In such investigations tritiated thymidine is often cautiously used to mark cells that are synthesizing DNA. Autoradiographic methodology makes possible the visualization of such cells, and it is possible to deduce the duration of DN,4 synthesis (S), mitosis (M), and the pre- and post-DNA synthetic interphase period (Gl and G2) from properly constructed experiments. The S period has been found to be fairly constant, usually from 6 to 8 h in duration [2, 31 in mammalian tissues, although a number of exceptions have been reported [18, 25, 431. This is true for in vivo and in vitro systems and for some rapidly proliferating embryonic tissues; furthermore, it is independent of cell generation time or species. A relatively constant but somewhat shorter (5-6 h) S period has been noted in bird somatic cells [2]. Inasmuch as the DNA content of both mammalian and avian cells is within a relatively constant range [32], Cameron and Greulich [3] and others suggested that the actual quantity of DNA might be a controlling factor of the temporal requirements for DNA synthesis. Tests of this hypothesis have produced conflicting results: There are, on the one hand, reports of a correlation between DNA content and the duration of DNA synthesis in both plant and animal tissues-nuclei having more DNA require more time for synthesis than those with less [9, 12, 23, 301. On the other hand, some workers have published results that seem to show an independence of the duration of the S period and the nuclear DNA content [4, 8, 9, 13, 21, 28, 311. 1 This investigation was supported by Grant no. GM 11702 and GM 8465 from the National Institutes of Health. a Present address: Department of Medicine, Division of Medical Genetics, University of Washington, Seattle, Wash. 98105, USA. 10 - 681813
Experimental
Cell Research
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Curia
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R. Troy
and
D. E. Wimber
In an attempt to clarify the situation, the present investigation of S period durations was conducted on the root tip cells of diploid and autotetraploid plants of Tradescuntia (spiderwort), Lycopersicum (tomato), Ehrharta (pasture grass), Ornithogrrlum (Star of Bethlehem), and Cymbidium (an orchid) in order to determine whether the duration of DNA synthesis is a function of DNA content, that is, whether a doubling of genetically identical DNA would correspondingly double the time required for DNA synthesis in a single species. \Ve have found in each set of comparisons that a relatively constant S period is either established or at least indicated by preliminary data. MATERIALS
AND
METHODS
Double labeling theory.-The duration of the DNA synthetic period in higher organisms has been measured utilizing several autoradiographic techniques [36]; the most direct and least laborious to date has been the double labeling technique developed by Wimber and Quastler [40] and Pilgrim and Maurer [22]. By exposing an actively proliferating population to a labeled DNA precursor (i.e., 3HTdR) for a brief time, all nuclei engaged in DNA synthesis incorporate label. If the population continues growth in the absenceof label for a period of time (t,), and is then labeled with I%-thymidine, before any of the previously labeled cells have passedthrough mitosis, the duration of the DNA synthetic period (f,) can be computed from the following relationship:
where NaHrefers to the number of nuclei labeled with 3H only, and K13cto all those labeled with %. The two labels are distinguishable becauseof the different energies of the beta-rays from 3H and 14C.The beta-particles from SH have an average track length of about one micron and thus produce silver grains localized in one plane in the photographic emulsion directly over the nucleus, while %-beta-particles travel an average distance of about 50 ,Ufrom the source, creating a spray of silver grains around and in many planes above the nucleus. Plant materials.-The following plants were used in this investigation: diploid and autotetraploid Tradescantia paludosa, Ehrharta erecta, Ornithogalum virens, Lycopersicum esculentum cv. San Marzano, diploid, triploid, and tetraploid Tulbaghia violacea, and Chrysanthemum corymbosum, C. yezoense, C. arcticum, and C. lacustre with 18, 56, -64, and 198 chromosomesrespectively. In addition, a tissue cultured Cymbidium clone (C. x Greenwood X C. x Green Giant) and its colchicine induced autotetraploid [41] were compared. The Ornithogalum autotetraploid arosespontaneously in a greenhouseculture; all others were produced by treating seedswith colchitine. The 3N Tulbaghia was obtained by crossinga diploid with a colchicine-induced tetraploid. Double labeling.-The plants were bare-rooted and grown in aerated, full-strength, Hoagland’s solution in a growth chamber under constant light and temperature (23 kl”C) for at least three weeks prior to and during the experiment. Plants were Experimental
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Constant
S period
in diploid-polyploid
plants
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isotopically labeled individually for 30 min in a temperature-equilibrated, aerated solution containing 1 @Z/ml 3HTdR (sp. act. 67 c/mM), rinsed for 4 min in running water, sampled, and returned to non-isotopic Hoagland’s solution to continue growth. After 2, 4 and 6 h, except with Lycopersicum which was for 5, 7 and 9 h, individual roots were removed and labeled for 30 min in 1, 2, or 3 PC/ml W-thymidine (sp. act. 27 mc/mM) and fixed immediately in 3 parts 100 per cent ethanol: 1 part glacial acetic acid. Diploid and tetraploid derivatives of the Cymbidium clone were cultured by a shoot meristem isolation technique [37]. They proliferated in a nutrient solution, forming masses of protocorm-like tissue. Fresh nutrient was supplied two weeks before the experiment was run. The cultures were maintained under constant light and temperature (23 +i”C) for one week prior to the experiment. “Conditioned” nutrient containing 1 /Lc/rnl $HTdR was poured over four cultures of each ploidy level for a 30 min labeling period. Each flask was carefully rinsed with two changes of unlabeled culture medium before adding unlabeled “conditioned” medium. After 4 and 6 h, two culin 10 ml beakers. tures of each ploidy level were labeled with 1 /AC/ml %-thymidine It was necessary to cut the tissue up somewhat at this time to fit it into the beakers. After 30 min the tissue was rinsed, separated into roots, shoots, and protocorm, and fixed as described for free living forms. The isotopes were obtained from New England Nuclear Corporation and International Chemical and Nuclear Corporation. It has been well established that the 3H-beta-ray deposits a great deal of energy in the chromosome, causing breakage [33] and perturbations to the normal population kinetics [38, 401. For this reason it is desirable to keep the levels of tritium incorporation as low as possible when followed by continued growth; this, however, necessitates rather long exposures of the autoradiographs. The low levels of isotope concentration chosen for these experiments were close to the limit of workability. AutoradiograpQPThe root tips were hydrolyzed for 12 min (15 min for Cymbidium) in 1 N HCl at 60X, Feulgen stained for l-2 h, and squashed on “subbed” slides in 45 per cent acetic acid. The siliceous cell walls of Ehrharta were digested in 4-5 per cent pectinase for 1 h after staining [42]. The slides were frozen on dry ice 151, placed in 100 per cent ethanol after coverslip removal, and gradually hydrated. A double layer of Kodak liquid NTB emulsion was applied to the slides by the dipping technique [17]. They were stored in the presence of Drierite (anhydrous calcium sulfate) at - 10°C for exposure periods of 3 to 6 months. Long exposure periods were deemed desirable in order to provide for the greatest possible ratio of nuclear to background label. After a 2-min development in Kodak Dektol, the slides were fixed, rinsed, and passed through the alcohol series to 100 per cent ethanol. Coverslips were mounted in Euparal. Autoradiographs were scored for 3H-only-labeled interphase cells, all X-labeled interphase cells, unlabeled interphases, labeled mitoses, and unlabeled mitoses (300 labeled cells per slide). Doubly labeled nuclei were not scored as such, since they are not distinguishable from those labeled with I% only. As a measure of proliferative activity and as a check on the stability of the duration of the S period, the labeling and mitotic indices were determined on slides of samples taken immediately after the initial isotope treatment and for every double labeled sample; populations greatly deviant from the mean were eliminated. Section.sPRoot tips of all free living plants except Lycopersicum, as well as diploid and tetraploid Bellevalia romana, were fixed in Craf III, embedded in paraffin, secEsperimenfal
CM
Research
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Maria
R. Troy and D. E. Wimber
tioned at a 10 p thickness, and stained with safranin fast green. Nuclear diameter (five slides or root tips, if available, with ten measurements selected from the largest to compensate for the plane of the section) were measured with a Zeiss ocular micrometer at 1250 x and the interphase nuclear volume calculated. The nucleus was assumed to be a perfect sphere for these estimates. Measured nuclei were located in the meristematic area of the root tips. RESULTS
AND
DISCUSSION
The mean S period durations, as determined by the double-labeling technique, are listed in Table 1. There is little variation between the diploidautotetraploid pairs of Tradescantia, Ornifhogalum, Lycopersicum, and the Cymbidium culture. The 10.7 and 10.4 h values obtained for 2K and 4N Tradescantia are quite close to previously reported values for diploid Tradescantia of 10.8 h utilizing the labeled-mitoses method [34] and 10.5 h with the double-labeling technique 1401. The mean S periods in diploid and tetraploid Lycopersicum are 7.2 and 7.6 h respectively, 7.1 and 7.7 h in the Cymbidium, and 7.9 h for both ploidy levels in Ornithogalum. In addition, preliminary data based on seven slides suggests an S period of about 6.1 h in diploid and 6.2 h in tetraploid Ehrharta root tip cells. The above values are probably slight over-estimates, since the populations remained in contact with the isotope for a 30-min labeling period, and this would tend to add a few minutes to our S period calculations. The means did not vary beyond 1 h in any of the compared pairs. In addition to intra-species comparisons, the S periods of related species (within the genus Chrysanthemum) were measured. The chrysanthemums have evolved into a large number of polyploid species in Europe and Asia with z = 9 in most of the species; some of the higher polyploids, however, are no longer exact multiples of nine [111. The range of ploidy is wide, extending to 22-ploid with 198 chromosomes. We found that the S periods of Chrysanthemum corymbosum 2N =18, C. yezoense 2N = 56, C. arcficum 2N = N 64, and C. lacustre 2N = 198, whose chromosome numbers varied ll-fold, were within a range of 4.8 to 6.3 h. Tanaka [28] has found that despite a size difference in the chromosomes of two related Chrysanthemum species, corresponding time differences were not required for DNA replication. Although the chromosomes of Chrysanthemum lineare are 1.5 to 2.0 times as long as those of C. nipponicum, the S periods of 10.5 and 9.5 h, respectively, were shown to be independent of chromosome length. These values, obtained from labeled mitoses curves, are not in line with the relative values obtained in our laboratory with the double-labeling Experimental
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Constant
S period
in diploid-polyploid
plants
149
technique. Whether these discrepancies result from technical inaccuracies, environmental differences, or reflect valid differences in the species’ temporal requirements for DNA synthesis is unknown at this time. The DNA content for particular species has been shown to be remarkably constant [16, 32]. In order to compare the DKA values of the plants used in 1. S period
TABLE
Plant
Tradescantia paludosa Ornithoqatum virens Cymbidium Lycopersicum esculentum Chrysanthemum corymbosum yezoense arcticum lacustre
Ploidy level and chromosome number
2N=12
duration
period 7 t,=Z
10.1
in higher Mean duration
plants
S (h k s.E.)
S period sample mean
j
t,=4
h
t, =
h
6 11
(h)
kO.8
(9)
10.8
kO.5
(8)
11.3iO.7
(8)
10.7
9.8 kO.3
(7)
10.6
kO.9
(7)
11.0+0.6
(9)
10.4
8.7 k 0.2
(6)
7.5 kO.2
(6)
7.7 kO.2
(11)
7.9
7.5 + 1.3
(4)
7.lkO.l
(4)
9.2 F0.4
(4)
7.9
2N=40
6.2 kO.1
(12)
7.9&0.4
(11)
7.1
4N=80
7.8 kO.3
(10)
7.5 * 0.9
(3)
7.7
45=24 2N=
6
4IV=12
2X-24
7.0 kO.4
(5)U
6.4 kO.9
(5)b
x.8+1.0
(5)C
7.2
-IN = 48
8.2 +0.4
(4)U
6.3 j0.2
(5)b
8.6 iO.4
(5)’
7.6
5.9
2N=18
6.6 2 0.3
(3)
5.1 kO.8
(6)
2N=56
4.5 & 0.1
(9)
6.1 +0.6
(8)
6.3 iO.4
(8)
-64
5.0 +0.9
(8)
4.3 +0.6
(7)
6.0 kO.6
(9)
5.1
= 198
6.7 kO.7
(7)
5.7 +0.3
(5)
6.4 kO.7
(10)
6.3
2N= 2x
Figure in parentheses refers to the a t, = 5 11; b t, = 7 11; ’ t, = 9 h.
number
4.8
of slides/sample.
this investigation, interphase nuclear volume calculations were made from sectioned material. Sparrow and Miksche [26] have reported a direct relationship between the interphase nuclear volume (in root tips treated for sectioning as described here) and DNA content as determined by chemical analysis. Although a number of exceptions to the relationship have been observed [l, 19, 241, it is probably satisfactory for the general comparisons made here. The interphase nuclear volume figures obtained for the tetraploid Relleoalicr, Ehrharta, Ornithogalum, Tradescantia, and Tulbaghia root tip nuclei (see Table 2) were found to be quite close to twice the diploid figures, as might be predicted. Since the deviations from expectation are not greater than 10 per cent, this would indicate that these rough nuclear measurements provide a fairly awurate assessment of relative DNA content. Experimental
Cell Research
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Maria
R. Troy
and
D. E. Wimber
Within the chrysanthemums an increase in chromosome number is often associated with diminishing chromosome size, which may indicate that evolution by polyploidization in the genus took place in the distant past; through the years loss of some of the redundant genetic information may have occurred. The nuclear volume calculations (Table 2) are in agreement with visual observations of decreasing chromosome size in the higher ploidy levels of these species: the nuclear volumes of C. yezoense, C. arcticum, and C. lacusfre are only 1.9, 1.8, and 8.0 times larger than the diploid, although the chromosome numbers have increased by factors of 3.0, 3.5, and 11.0. Although the nuclear volume does not increase in proportion to the ploidy level, there is no question that the quantity of DNA per nucleus does rise to some extent with increase in ploidy. Casual observation of Feulgen stained material easily substantiates this. Technical and experimental difficulties with labeling the diploid, triploid, and tetraploid Tulbaghia clones resulted in no reliable double-labeling data from this group. However, gross examination of autoradiographic preparations from roots exposed to 3HTdR alone, demonstrates a proportionately greater uptake with increasing chromosome number. The following average grain counts were observed over 20 labeled nuclei from root tips which had been treated with 5 PC/ml 3HTdR for 30 min, fixed immediately, and exposed for one and one-half weeks: 2N- 16 k 4; 3N- 28 + 6; and 4Pi - 48 k24 ( k standard deviation). The number of silver grains over such material is generally considered to reflect metabolic rate. These increasing grain counts are in support of the general findings of this study that higher ploidy levels of a species synthesize at a greater average rate and probably do not require more time to synthesize more DNA. Das and Alfert [7] have reported a 50 per cent increase in the rate of DNA synthesis in autotetraploid Antirrhinum majus (snapdragon), compared with the diploid, as determined by grain count analysis. They observed that this increase is more closely correlated with the tetraploid nuclear surface area than nuclear volume. But, on the other hand, similar labeled mitoses curves \\-ere noted in both ploidy levels of the same material [6]. The interval during which at least 50 per cent of the mitoses are labeled, generally considered to equal the S period, is 4 h for each. ,4lthough the S period and rate data are not in complete agreement, the S period is not a function of DNA content, and the average nuclear rate of DNA synthesis does increase to some extent \vith increase in ploidy. Unequal nuclear rates of DNL4 synthesis during the S period have been demonstrated in a number of tissues [lo, 14, 201. Assuming a constant moleExperimental
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Constant
S period
in diploid-polyploid
plants
151
cular rate of DNA synthesis under constant conditions, the average nuclear rate would be expected to vary with the number of units of replication, or “replicons”, demonstrating uptake at any one moment during S. In addition, replication pattern studies have shown a specific, temporal pattern of DNA synthesis throughout the complement [27, 29, 35’. Generally, homologous TABLE 2. Interphase
Plant Bhrharta erecta Ornithogalum vii-ens Belleualia romana Tulbaghia oiolacea Tradescantia pahcdosa Chrysanthemum corymbosnm nipponica yezoense arcticum lacustre
nuclear Ploidy level
volume measurements
Chromosome number
Interphase nuclear volume, p””
2N 1s 2N 4s 2s 4N 2N 3N 4N 2N -1s
21 48 6 12 8 16 12 18 21 12 24
196.0 392.0 194.1 1001.0 625.3 1320.6 1025.4 1633.9 2150.1 1050.2 2150.1
2N 2N 2x 2iY 2x
18 18 56 - 6-l 198
525.0 575.4 1001.0 953.3 4200.0
sites synthesize DNA at the same time, although distinct differences in S period timing patterns have been reported within certain homologous pairs in mammalian tissues [ 151. Thus, with a doubling of ploidy one might predict that the temporal pattern of DNA replication would remain the same with corresponding portions of homologues replicating simultaneously, and the pattern of replication throughout the complement being retained. Such a chromosome doubling \vould not be expected to lengthen the time required for DNA synthesis provided that sufficient precursors and enzymes were present so as not to limit synthetic rates. This would cause a doubling of the average nuclear rate of DNA synthesis, and this is precisely what has been demonstrated here in the five diploid-tetraploid pairs. Further support for Experimental
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Maria
R. Troy
and D. E. Wimber
this hypothesis
is gleaned from the fairly similar S periods among the Chrysanthemum species whose increased chromosome numbers probably represent approximate genomic increases. The results presented here are not in agreement with those of Van’t Hof 1301 which pointed to a direct relationship between the DNA content, S period duration, and the average rate of DNA synthesis. Although it may be demonstrated that in distantly related plants, an increase in DNA per cell may be accompanied by an increased S period duration, the relationship has not been shown to hold among related plants nor among various ploidy levels within a single species. It may be noted that among the seven plants from which Van’t Hof drew his conclusions, there were three species of Allium whose S periods increased only slightly with, and not in proportion to, increased DNA content. A possible difference between V’an’t Hof’s experiments and our own is in his use of roots from germinating seeds and our use of roots from photosynthesizing plants. In both our works, Lycopersicum esculentum was used (though possibly different varieties), and the results were different. Van’t Hof found an S period of 4.3 h, while we found one of about 7 h. Similarly, in Crepis capillaris Wimber [39] found an S period of 7.5 h in roots taken from a photosynthesizing plant, while Van’t Hof found an S period of 3.25 h in germinating seeds. It is possible that these dit‘ferences reflect inequalities in nutritional states of the cells or changes somehow modulated by different functional conditions of the cells at various times in the life cycle of the organism. These possibilities merit further attention.
SUMMARY
The duration of the DNA synthetic (S) period is shown to be relatively constant, thus independent of DNA content, in diploid-autotetraploid comparisons in several higher plants and among several species of the genus, Chrysanthemum. An 3H-, 14C-thymidine double-labeling technique was used for the measurements. The following mean S periods were obtained for diploid and autotetraploid plants respectively: 7.2 and 7.6 h for Lycopersicum esculentum, 10.7 and 10.4 h for Tradescantia paludosa, 7.9 and 7.9 h for Ornithogalum virens, 7.1 and 7.7 h for a Cymitbium in culture and 6.1 and 6.2 h from preliminary data for Ehrharta erecta. In the Chrysanthemum species, the mean S periods were 5.9 h in C. corymbosum 2N = 18, 4.8 in C. yezoense 2N = 56, 5.1 in C. arcticum 2N = 64 and 6.3 h in C. lacustre 2N = 198. These results point to a linear increase in the average nuclear rate of DNA Experimental
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53
Constant
S period
in diploid-polyploid
153
plants
synthesis with increasing ploidy level and this is further supported b;\ findings of progressively increased grain counts over 2N, 3N and 4N Tulbnghia rGolacea nuclei. Replication pattern studies by others have noted a specific, temporal pattern of DNA synthesis throughout the chromosomal complement; thus, one would not expect an increased S period duration to result from an increase in ploidy. The results presented here are not in agreement with those of Van’t HOP w-hich indicated a direct relationship between DS,1 content and S period duration in a number of unrelated higher plants; however, differences in experimental conditions may account in part for the discrepancies. We are grateful to Professor plants and to Dr A. H. Sparrow mums.
G. L. Stebbins for the Lycopersicum and Ehrharfa for the Omithogalum, Trndescantin and Chrysanthe-
REFERENCES I.
BAETCKE, K. P., SPARROW, A. H., NALXAN, C. H. and SCHMXMMBR, S. S., Proc. sci. us 58, 533 (1967). CAMERON, I. L., J. Cell Biol. 20, 185 (1964). CAMERON, I. L. and GREULICH, R. C., J. Cell Biol. 18, 31 (1963). CAMERON, I. L. and STONE, G. E., Expfl Cell Res. 36, 510 (1964). CONGER, A. D. and FAIRCHILD, L. &I., Stain Technol. 28, 281 (1953). DAS, N. K., Unpublished. DAS, N. K. and ALFERT, M., 2nd Ann. Meeting, Am. Sot. Cell Biol. p. 41 1962. DEFENDI, V. and MANSON, L. A., Pathol. Biol. Semaine Hop. 9, 525 (1961).
Nat1 Acad
2. 3. 4. 5. 6. 7. 8. 9. ~ Nature 198, 359 (1963). 10. DENDY, P. P. and CLEAVER, J. E., Intern. J. Radialion Biol. 8, 301 (1964). 11. DOWRICK, G. J., Heredity 6, 365 (1952). 12. GOLDFEDER, A., Nature 207, 612 (1965). 13. GRAHAM, C. F., Expfl Cell Res. 43, 13 (1966). 14. HOWARD, A. and DEWEY, D. L., Exptl Cell Res. 24, 623 (1961). 15. Hsu, T. C., SCHMID, W. and STUBBLEFIELD,E., in M. LocKE(~~.), The Role of Chromosomes in Development, p. 83. Academic Press, New York, 1964. 16. JAMES, J., Cyfogenetics 4, 19 (1965). 17. MESSIER, B. and LEBLOND, C. P., Proc. Sot. Erptl Biol. Med. 96, 7 (1957). 18. MIKULICICH, A. G. and YOUNG, R. W., Inuesf. Ophfhalmol. 2, 344 (1963). 19. MIKSCHE, J. P., Bull. Torrey Bofan. Club 92, 1 (1965). 20. NEWTON, A. A. and WILDY, P., Expfl Cell Res. 16, 624 (1959). 21. OEHLERT, W., SEEMAYER, N. and LAUF, P., Beitr. Pathol. Anat. Allgem. Pathol. 127, 63 (1962). 22. PILGRIM, CH. and MAURER, W., Nafurwiss. 49, 544 (1962). 23. PRASAD, A. B. and GODWARD, hI. B. E., Rad. Bof. 5, 465 (1965). 24. SCHRADER, F.and LEUCHTENBERGER, C., Exptl Cell Res. 1,421(1950). 25. SHERMAN, F. G. QUASTLER, H. and WIMBER, D. R., Exptl Cell Res. 25, 114 (1961). 26. SPARROW, A. H. and MIKSCHE, J. P., Science 134, 282 (1961). 27. TANAKA, R., Botan. Mag., Tokyo 78, 50 (1965). 28. ibid. 79, 447 (1966). 29. TAYLOR, J. fi., J. Biophys. Biochem. Cytol. 7, 455 (1960). 30. VAN’T HOF, J. Expfl Cell Res. 39, 48 (1965). 31. --ibid. 41, 274 (1966).
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32. VENDRELY, R., in E. CHARGAFF and J. N. DAVIDSOK (eds), The Nucleic Acids, vol. 2, p. 155. Academic Press, New York, 1955. 33. WIMBER, D. E., Proc. Natl Acad. Set. US 45, 839 (1959). 34. -iim. J. Bot. 47, 828 (1960). 35. ~Exptl Cell Res. 23, 402 (1961). 36. --in L. F. LAMERTOX and R. J. M. FRY (eds), Cell Proliferation, p. 1. Blackwell, Oxford 1963. 37. ~Am. Orchid Sot. BuU. 32, IO5 (1963). 38. -.in L. G. AUGENSTEIN, R. MASON and H. QUASTLER (eds), Advances in Radiation Biology, vd, 1, p. 85. Academic Press, New York, 1964. 39. -~Unpublished. 40. WIMBER, D. E. and QUASTLER, H., Exptl Cell Res. 30, 8 (1963). 41. WwBER, D. E. and VAN COTT, A., in L. R. DEGARMO (ed.), Proc. Fifth World Orchid Conference, p. 25, 1966. American Orchid Society. 42. W’OLFF, S. and LUIPPOLD, H. E., Stain Technol. 31, 201 (1956). 43. WOLFSBERG, 111. F. Ezl’tZ Cell Res. 35, 119 (1964).
Experimental
Cell
Research
53
by i\catiemic
Experimental
Press Inc.
Cell Research
53, 145-154
(1968)
l-15
EVIDENCE FOR A CONSTANCY OF THE DNA SYNTHETIC PERIOD BETWEEN DIPLOID-POLYPLOID GROUPS IN PLANTS1 MARIA Biology
Department,
R. TROY2 University Received
and
D. E. WIMBER
of Oregon, March
Eugene,
Oreg.
97403,
USA
18, 1968
THE relative durations of the subdivisions of the nuclear cycle have been studied intensively in recent years in a large number of organisms. In such investigations tritiated thymidine is often cautiously used to mark cells that are synthesizing DNA. Autoradiographic methodology makes possible the visualization of such cells, and it is possible to deduce the duration of DN,4 synthesis (S), mitosis (M), and the pre- and post-DNA synthetic interphase period (Gl and G2) from properly constructed experiments. The S period has been found to be fairly constant, usually from 6 to 8 h in duration [2, 31 in mammalian tissues, although a number of exceptions have been reported [18, 25, 431. This is true for in vivo and in vitro systems and for some rapidly proliferating embryonic tissues; furthermore, it is independent of cell generation time or species. A relatively constant but somewhat shorter (5-6 h) S period has been noted in bird somatic cells [2]. Inasmuch as the DNA content of both mammalian and avian cells is within a relatively constant range [32], Cameron and Greulich [3] and others suggested that the actual quantity of DNA might be a controlling factor of the temporal requirements for DNA synthesis. Tests of this hypothesis have produced conflicting results: There are, on the one hand, reports of a correlation between DNA content and the duration of DNA synthesis in both plant and animal tissues-nuclei having more DNA require more time for synthesis than those with less [9, 12, 23, 301. On the other hand, some workers have published results that seem to show an independence of the duration of the S period and the nuclear DNA content [4, 8, 9, 13, 21, 28, 311. 1 This investigation was supported by Grant no. GM 11702 and GM 8465 from the National Institutes of Health. a Present address: Department of Medicine, Division of Medical Genetics, University of Washington, Seattle, Wash. 98105, USA. 10 - 681813
Experimental
Cell Research
53
Curia
146
R. Troy
and
D. E. Wimber
In an attempt to clarify the situation, the present investigation of S period durations was conducted on the root tip cells of diploid and autotetraploid plants of Tradescuntia (spiderwort), Lycopersicum (tomato), Ehrharta (pasture grass), Ornithogrrlum (Star of Bethlehem), and Cymbidium (an orchid) in order to determine whether the duration of DNA synthesis is a function of DNA content, that is, whether a doubling of genetically identical DNA would correspondingly double the time required for DNA synthesis in a single species. \Ve have found in each set of comparisons that a relatively constant S period is either established or at least indicated by preliminary data. MATERIALS
AND
METHODS
Double labeling theory.-The duration of the DNA synthetic period in higher organisms has been measured utilizing several autoradiographic techniques [36]; the most direct and least laborious to date has been the double labeling technique developed by Wimber and Quastler [40] and Pilgrim and Maurer [22]. By exposing an actively proliferating population to a labeled DNA precursor (i.e., 3HTdR) for a brief time, all nuclei engaged in DNA synthesis incorporate label. If the population continues growth in the absenceof label for a period of time (t,), and is then labeled with I%-thymidine, before any of the previously labeled cells have passedthrough mitosis, the duration of the DNA synthetic period (f,) can be computed from the following relationship:
where NaHrefers to the number of nuclei labeled with 3H only, and K13cto all those labeled with %. The two labels are distinguishable becauseof the different energies of the beta-rays from 3H and 14C.The beta-particles from SH have an average track length of about one micron and thus produce silver grains localized in one plane in the photographic emulsion directly over the nucleus, while %-beta-particles travel an average distance of about 50 ,Ufrom the source, creating a spray of silver grains around and in many planes above the nucleus. Plant materials.-The following plants were used in this investigation: diploid and autotetraploid Tradescantia paludosa, Ehrharta erecta, Ornithogalum virens, Lycopersicum esculentum cv. San Marzano, diploid, triploid, and tetraploid Tulbaghia violacea, and Chrysanthemum corymbosum, C. yezoense, C. arcticum, and C. lacustre with 18, 56, -64, and 198 chromosomesrespectively. In addition, a tissue cultured Cymbidium clone (C. x Greenwood X C. x Green Giant) and its colchicine induced autotetraploid [41] were compared. The Ornithogalum autotetraploid arosespontaneously in a greenhouseculture; all others were produced by treating seedswith colchitine. The 3N Tulbaghia was obtained by crossinga diploid with a colchicine-induced tetraploid. Double labeling.-The plants were bare-rooted and grown in aerated, full-strength, Hoagland’s solution in a growth chamber under constant light and temperature (23 kl”C) for at least three weeks prior to and during the experiment. Plants were Experimental
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Constant
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isotopically labeled individually for 30 min in a temperature-equilibrated, aerated solution containing 1 @Z/ml 3HTdR (sp. act. 67 c/mM), rinsed for 4 min in running water, sampled, and returned to non-isotopic Hoagland’s solution to continue growth. After 2, 4 and 6 h, except with Lycopersicum which was for 5, 7 and 9 h, individual roots were removed and labeled for 30 min in 1, 2, or 3 PC/ml W-thymidine (sp. act. 27 mc/mM) and fixed immediately in 3 parts 100 per cent ethanol: 1 part glacial acetic acid. Diploid and tetraploid derivatives of the Cymbidium clone were cultured by a shoot meristem isolation technique [37]. They proliferated in a nutrient solution, forming masses of protocorm-like tissue. Fresh nutrient was supplied two weeks before the experiment was run. The cultures were maintained under constant light and temperature (23 +i”C) for one week prior to the experiment. “Conditioned” nutrient containing 1 /Lc/rnl $HTdR was poured over four cultures of each ploidy level for a 30 min labeling period. Each flask was carefully rinsed with two changes of unlabeled culture medium before adding unlabeled “conditioned” medium. After 4 and 6 h, two culin 10 ml beakers. tures of each ploidy level were labeled with 1 /AC/ml %-thymidine It was necessary to cut the tissue up somewhat at this time to fit it into the beakers. After 30 min the tissue was rinsed, separated into roots, shoots, and protocorm, and fixed as described for free living forms. The isotopes were obtained from New England Nuclear Corporation and International Chemical and Nuclear Corporation. It has been well established that the 3H-beta-ray deposits a great deal of energy in the chromosome, causing breakage [33] and perturbations to the normal population kinetics [38, 401. For this reason it is desirable to keep the levels of tritium incorporation as low as possible when followed by continued growth; this, however, necessitates rather long exposures of the autoradiographs. The low levels of isotope concentration chosen for these experiments were close to the limit of workability. AutoradiograpQPThe root tips were hydrolyzed for 12 min (15 min for Cymbidium) in 1 N HCl at 60X, Feulgen stained for l-2 h, and squashed on “subbed” slides in 45 per cent acetic acid. The siliceous cell walls of Ehrharta were digested in 4-5 per cent pectinase for 1 h after staining [42]. The slides were frozen on dry ice 151, placed in 100 per cent ethanol after coverslip removal, and gradually hydrated. A double layer of Kodak liquid NTB emulsion was applied to the slides by the dipping technique [17]. They were stored in the presence of Drierite (anhydrous calcium sulfate) at - 10°C for exposure periods of 3 to 6 months. Long exposure periods were deemed desirable in order to provide for the greatest possible ratio of nuclear to background label. After a 2-min development in Kodak Dektol, the slides were fixed, rinsed, and passed through the alcohol series to 100 per cent ethanol. Coverslips were mounted in Euparal. Autoradiographs were scored for 3H-only-labeled interphase cells, all X-labeled interphase cells, unlabeled interphases, labeled mitoses, and unlabeled mitoses (300 labeled cells per slide). Doubly labeled nuclei were not scored as such, since they are not distinguishable from those labeled with I% only. As a measure of proliferative activity and as a check on the stability of the duration of the S period, the labeling and mitotic indices were determined on slides of samples taken immediately after the initial isotope treatment and for every double labeled sample; populations greatly deviant from the mean were eliminated. Section.sPRoot tips of all free living plants except Lycopersicum, as well as diploid and tetraploid Bellevalia romana, were fixed in Craf III, embedded in paraffin, secEsperimenfal
CM
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Maria
R. Troy and D. E. Wimber
tioned at a 10 p thickness, and stained with safranin fast green. Nuclear diameter (five slides or root tips, if available, with ten measurements selected from the largest to compensate for the plane of the section) were measured with a Zeiss ocular micrometer at 1250 x and the interphase nuclear volume calculated. The nucleus was assumed to be a perfect sphere for these estimates. Measured nuclei were located in the meristematic area of the root tips. RESULTS
AND
DISCUSSION
The mean S period durations, as determined by the double-labeling technique, are listed in Table 1. There is little variation between the diploidautotetraploid pairs of Tradescantia, Ornifhogalum, Lycopersicum, and the Cymbidium culture. The 10.7 and 10.4 h values obtained for 2K and 4N Tradescantia are quite close to previously reported values for diploid Tradescantia of 10.8 h utilizing the labeled-mitoses method [34] and 10.5 h with the double-labeling technique 1401. The mean S periods in diploid and tetraploid Lycopersicum are 7.2 and 7.6 h respectively, 7.1 and 7.7 h in the Cymbidium, and 7.9 h for both ploidy levels in Ornithogalum. In addition, preliminary data based on seven slides suggests an S period of about 6.1 h in diploid and 6.2 h in tetraploid Ehrharta root tip cells. The above values are probably slight over-estimates, since the populations remained in contact with the isotope for a 30-min labeling period, and this would tend to add a few minutes to our S period calculations. The means did not vary beyond 1 h in any of the compared pairs. In addition to intra-species comparisons, the S periods of related species (within the genus Chrysanthemum) were measured. The chrysanthemums have evolved into a large number of polyploid species in Europe and Asia with z = 9 in most of the species; some of the higher polyploids, however, are no longer exact multiples of nine [111. The range of ploidy is wide, extending to 22-ploid with 198 chromosomes. We found that the S periods of Chrysanthemum corymbosum 2N =18, C. yezoense 2N = 56, C. arcficum 2N = N 64, and C. lacustre 2N = 198, whose chromosome numbers varied ll-fold, were within a range of 4.8 to 6.3 h. Tanaka [28] has found that despite a size difference in the chromosomes of two related Chrysanthemum species, corresponding time differences were not required for DNA replication. Although the chromosomes of Chrysanthemum lineare are 1.5 to 2.0 times as long as those of C. nipponicum, the S periods of 10.5 and 9.5 h, respectively, were shown to be independent of chromosome length. These values, obtained from labeled mitoses curves, are not in line with the relative values obtained in our laboratory with the double-labeling Experimental
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149
technique. Whether these discrepancies result from technical inaccuracies, environmental differences, or reflect valid differences in the species’ temporal requirements for DNA synthesis is unknown at this time. The DNA content for particular species has been shown to be remarkably constant [16, 32]. In order to compare the DKA values of the plants used in 1. S period
TABLE
Plant
Tradescantia paludosa Ornithoqatum virens Cymbidium Lycopersicum esculentum Chrysanthemum corymbosum yezoense arcticum lacustre
Ploidy level and chromosome number
2N=12
duration
period 7 t,=Z
10.1
in higher Mean duration
plants
S (h k s.E.)
S period sample mean
j
t,=4
h
t, =
h
6 11
(h)
kO.8
(9)
10.8
kO.5
(8)
11.3iO.7
(8)
10.7
9.8 kO.3
(7)
10.6
kO.9
(7)
11.0+0.6
(9)
10.4
8.7 k 0.2
(6)
7.5 kO.2
(6)
7.7 kO.2
(11)
7.9
7.5 + 1.3
(4)
7.lkO.l
(4)
9.2 F0.4
(4)
7.9
2N=40
6.2 kO.1
(12)
7.9&0.4
(11)
7.1
4N=80
7.8 kO.3
(10)
7.5 * 0.9
(3)
7.7
45=24 2N=
6
4IV=12
2X-24
7.0 kO.4
(5)U
6.4 kO.9
(5)b
x.8+1.0
(5)C
7.2
-IN = 48
8.2 +0.4
(4)U
6.3 j0.2
(5)b
8.6 iO.4
(5)’
7.6
5.9
2N=18
6.6 2 0.3
(3)
5.1 kO.8
(6)
2N=56
4.5 & 0.1
(9)
6.1 +0.6
(8)
6.3 iO.4
(8)
-64
5.0 +0.9
(8)
4.3 +0.6
(7)
6.0 kO.6
(9)
5.1
= 198
6.7 kO.7
(7)
5.7 +0.3
(5)
6.4 kO.7
(10)
6.3
2N= 2x
Figure in parentheses refers to the a t, = 5 11; b t, = 7 11; ’ t, = 9 h.
number
4.8
of slides/sample.
this investigation, interphase nuclear volume calculations were made from sectioned material. Sparrow and Miksche [26] have reported a direct relationship between the interphase nuclear volume (in root tips treated for sectioning as described here) and DNA content as determined by chemical analysis. Although a number of exceptions to the relationship have been observed [l, 19, 241, it is probably satisfactory for the general comparisons made here. The interphase nuclear volume figures obtained for the tetraploid Relleoalicr, Ehrharta, Ornithogalum, Tradescantia, and Tulbaghia root tip nuclei (see Table 2) were found to be quite close to twice the diploid figures, as might be predicted. Since the deviations from expectation are not greater than 10 per cent, this would indicate that these rough nuclear measurements provide a fairly awurate assessment of relative DNA content. Experimental
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R. Troy
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D. E. Wimber
Within the chrysanthemums an increase in chromosome number is often associated with diminishing chromosome size, which may indicate that evolution by polyploidization in the genus took place in the distant past; through the years loss of some of the redundant genetic information may have occurred. The nuclear volume calculations (Table 2) are in agreement with visual observations of decreasing chromosome size in the higher ploidy levels of these species: the nuclear volumes of C. yezoense, C. arcticum, and C. lacusfre are only 1.9, 1.8, and 8.0 times larger than the diploid, although the chromosome numbers have increased by factors of 3.0, 3.5, and 11.0. Although the nuclear volume does not increase in proportion to the ploidy level, there is no question that the quantity of DNA per nucleus does rise to some extent with increase in ploidy. Casual observation of Feulgen stained material easily substantiates this. Technical and experimental difficulties with labeling the diploid, triploid, and tetraploid Tulbaghia clones resulted in no reliable double-labeling data from this group. However, gross examination of autoradiographic preparations from roots exposed to 3HTdR alone, demonstrates a proportionately greater uptake with increasing chromosome number. The following average grain counts were observed over 20 labeled nuclei from root tips which had been treated with 5 PC/ml 3HTdR for 30 min, fixed immediately, and exposed for one and one-half weeks: 2N- 16 k 4; 3N- 28 + 6; and 4Pi - 48 k24 ( k standard deviation). The number of silver grains over such material is generally considered to reflect metabolic rate. These increasing grain counts are in support of the general findings of this study that higher ploidy levels of a species synthesize at a greater average rate and probably do not require more time to synthesize more DNA. Das and Alfert [7] have reported a 50 per cent increase in the rate of DNA synthesis in autotetraploid Antirrhinum majus (snapdragon), compared with the diploid, as determined by grain count analysis. They observed that this increase is more closely correlated with the tetraploid nuclear surface area than nuclear volume. But, on the other hand, similar labeled mitoses curves \\-ere noted in both ploidy levels of the same material [6]. The interval during which at least 50 per cent of the mitoses are labeled, generally considered to equal the S period, is 4 h for each. ,4lthough the S period and rate data are not in complete agreement, the S period is not a function of DNA content, and the average nuclear rate of DNA synthesis does increase to some extent \vith increase in ploidy. Unequal nuclear rates of DNL4 synthesis during the S period have been demonstrated in a number of tissues [lo, 14, 201. Assuming a constant moleExperimental
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Constant
S period
in diploid-polyploid
plants
151
cular rate of DNA synthesis under constant conditions, the average nuclear rate would be expected to vary with the number of units of replication, or “replicons”, demonstrating uptake at any one moment during S. In addition, replication pattern studies have shown a specific, temporal pattern of DNA synthesis throughout the complement [27, 29, 35’. Generally, homologous TABLE 2. Interphase
Plant Bhrharta erecta Ornithogalum vii-ens Belleualia romana Tulbaghia oiolacea Tradescantia pahcdosa Chrysanthemum corymbosnm nipponica yezoense arcticum lacustre
nuclear Ploidy level
volume measurements
Chromosome number
Interphase nuclear volume, p””
2N 1s 2N 4s 2s 4N 2N 3N 4N 2N -1s
21 48 6 12 8 16 12 18 21 12 24
196.0 392.0 194.1 1001.0 625.3 1320.6 1025.4 1633.9 2150.1 1050.2 2150.1
2N 2N 2x 2iY 2x
18 18 56 - 6-l 198
525.0 575.4 1001.0 953.3 4200.0
sites synthesize DNA at the same time, although distinct differences in S period timing patterns have been reported within certain homologous pairs in mammalian tissues [ 151. Thus, with a doubling of ploidy one might predict that the temporal pattern of DNA replication would remain the same with corresponding portions of homologues replicating simultaneously, and the pattern of replication throughout the complement being retained. Such a chromosome doubling \vould not be expected to lengthen the time required for DNA synthesis provided that sufficient precursors and enzymes were present so as not to limit synthetic rates. This would cause a doubling of the average nuclear rate of DNA synthesis, and this is precisely what has been demonstrated here in the five diploid-tetraploid pairs. Further support for Experimental
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R. Troy
and D. E. Wimber
this hypothesis
is gleaned from the fairly similar S periods among the Chrysanthemum species whose increased chromosome numbers probably represent approximate genomic increases. The results presented here are not in agreement with those of Van’t Hof 1301 which pointed to a direct relationship between the DNA content, S period duration, and the average rate of DNA synthesis. Although it may be demonstrated that in distantly related plants, an increase in DNA per cell may be accompanied by an increased S period duration, the relationship has not been shown to hold among related plants nor among various ploidy levels within a single species. It may be noted that among the seven plants from which Van’t Hof drew his conclusions, there were three species of Allium whose S periods increased only slightly with, and not in proportion to, increased DNA content. A possible difference between V’an’t Hof’s experiments and our own is in his use of roots from germinating seeds and our use of roots from photosynthesizing plants. In both our works, Lycopersicum esculentum was used (though possibly different varieties), and the results were different. Van’t Hof found an S period of 4.3 h, while we found one of about 7 h. Similarly, in Crepis capillaris Wimber [39] found an S period of 7.5 h in roots taken from a photosynthesizing plant, while Van’t Hof found an S period of 3.25 h in germinating seeds. It is possible that these dit‘ferences reflect inequalities in nutritional states of the cells or changes somehow modulated by different functional conditions of the cells at various times in the life cycle of the organism. These possibilities merit further attention.
SUMMARY
The duration of the DNA synthetic (S) period is shown to be relatively constant, thus independent of DNA content, in diploid-autotetraploid comparisons in several higher plants and among several species of the genus, Chrysanthemum. An 3H-, 14C-thymidine double-labeling technique was used for the measurements. The following mean S periods were obtained for diploid and autotetraploid plants respectively: 7.2 and 7.6 h for Lycopersicum esculentum, 10.7 and 10.4 h for Tradescantia paludosa, 7.9 and 7.9 h for Ornithogalum virens, 7.1 and 7.7 h for a Cymitbium in culture and 6.1 and 6.2 h from preliminary data for Ehrharta erecta. In the Chrysanthemum species, the mean S periods were 5.9 h in C. corymbosum 2N = 18, 4.8 in C. yezoense 2N = 56, 5.1 in C. arcticum 2N = 64 and 6.3 h in C. lacustre 2N = 198. These results point to a linear increase in the average nuclear rate of DNA Experimental
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Constant
S period
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153
plants
synthesis with increasing ploidy level and this is further supported b;\ findings of progressively increased grain counts over 2N, 3N and 4N Tulbnghia rGolacea nuclei. Replication pattern studies by others have noted a specific, temporal pattern of DNA synthesis throughout the chromosomal complement; thus, one would not expect an increased S period duration to result from an increase in ploidy. The results presented here are not in agreement with those of Van’t HOP w-hich indicated a direct relationship between DS,1 content and S period duration in a number of unrelated higher plants; however, differences in experimental conditions may account in part for the discrepancies. We are grateful to Professor plants and to Dr A. H. Sparrow mums.
G. L. Stebbins for the Lycopersicum and Ehrharfa for the Omithogalum, Trndescantin and Chrysanthe-
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32. VENDRELY, R., in E. CHARGAFF and J. N. DAVIDSOK (eds), The Nucleic Acids, vol. 2, p. 155. Academic Press, New York, 1955. 33. WIMBER, D. E., Proc. Natl Acad. Set. US 45, 839 (1959). 34. -iim. J. Bot. 47, 828 (1960). 35. ~Exptl Cell Res. 23, 402 (1961). 36. --in L. F. LAMERTOX and R. J. M. FRY (eds), Cell Proliferation, p. 1. Blackwell, Oxford 1963. 37. ~Am. Orchid Sot. BuU. 32, IO5 (1963). 38. -.in L. G. AUGENSTEIN, R. MASON and H. QUASTLER (eds), Advances in Radiation Biology, vd, 1, p. 85. Academic Press, New York, 1964. 39. -~Unpublished. 40. WIMBER, D. E. and QUASTLER, H., Exptl Cell Res. 30, 8 (1963). 41. WwBER, D. E. and VAN COTT, A., in L. R. DEGARMO (ed.), Proc. Fifth World Orchid Conference, p. 25, 1966. American Orchid Society. 42. W’OLFF, S. and LUIPPOLD, H. E., Stain Technol. 31, 201 (1956). 43. WOLFSBERG, 111. F. Ezl’tZ Cell Res. 35, 119 (1964).
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