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Embryogenesis and ribosomal DNA in carrot cell suspensions cultured in vitro
Plant Science Letters, 33 (1984) 23--29
23
Elsevier Scientific Publishers Ireland Ltd.
EMBRYOGENESIS AND RIBOSOMAL DNA IN CARROT CELL SUSPENSIONS CULTURED IN VITRO
KIYOSHI MASUDA, YOSHIO KIKUTA and YOZO OKAZAWA
Faculty of Agriculture, Department of Botany, Hokkaido University, Sapporo, 060 (Japan) (Received January 31st, 1983) (Revision received July 13th, 1983) (Accepted July 13th, 1983)
SUMMARY
The contents of nucleic acids and rDNA were estimated during the development of carrot cell suspensions cultured under two different conditions. The cells transferred from stock culture to the medium without 2,4-dichlorophenoxyacetic acid (2,4-D) induced the embryogenesis (embryogenic culture~ while the cells inoculated to the medium with 0.2 mg/l 2,4-D did not form any embryos (non-embryogenic culture). The ratio of RNA to DNA of both cultures increased in the early stage of the culture. The rise of the ratio in embryogenic culture was much higher than that in non-embryogenic culture, which showed that embryogenic culture accumulated RNA prior to the formation of embryos. The rDNA amount of non-embryogenic culture remained constant throughout the culture period. Although embryogenic culture showed a slight change in rDNA amount, the differences were at most 12% and the quantitative stability of the rDNA was demonstrated during the development of carrot cell suspension cultures. K e y w o r d s : D a u c u s c a r o t a - - Somatic embryogenesis -- rDNA
INTRODUCTION
It has been well documented that somatic embryogenesis in carrot cell suspension is readily elicited when cells cultured in a medium supplemented with 2,4-D are subsequently transferred to an auxin-free medium [1,2]. It may be expected that changes of cell metabolism in culture would have to occur at an early stage of embryogenic culture. In fact, much attention Abbreviations: 2,4-D, 2,4-dichlorophenoxyacetic acid; SLS, sodium lauryl sulfate; SSC, standard sodium citrate buffer. 0304-4211/84/$03.00 © 1984 Elsevier Scientific Publishers Ireland Ltd. Printed and Published in Ireland
24
has been paid to embryogenesis in relation to a pronounced accumulation of RNA [5] and to a noticeable stimulation in chromatin~iirected RNA synthesis [3,4]. Although it is generally known that plant cells contain a large number of copies of rRNA genes, little information is presently available on the evidence for their differential replication [6]. For instance it has been reported that the changes of rDNA amount of flax plants seem to be closely associated with the phenotypical modification, which is induced by the alteration of nutritional and environmental conditions [7,8]. It may be anticipated, therefore, that the rRNA genes in plants would also be liable to change in advance of their development. In the present study, the question arises whether redifferentiation in plant cell cultures is linked with the quantitative change of rRNA genes. The cell suspension culture of carrot provides a suitable system for evaluation of this problem. In this paper, rDNA amounts in carrot cell suspensions were estimated during their developmental period, and the possibility was explored that RNA accumulation at the early stage of embryogenic culture would be associated with a quantitative variation of rRNA genes. MATERIALS AND METHODS
Culture The suspension cultures of carrot (Daucus carota L. cv. Red Core Chantenay) were established from sterile seedlings and maintained in liquid medium with 0.2 rag/1 2,4-D [2]. The inocula for embryogenic culture were prepared from 13-day-old stock culture. The cells passed through a 177-~m metal screen were collected on a 44-~m screen, washed with fresh medium and inoculated to medium without 2,4-D (--D culture). For non-embryogenic culture, the inocula were subcultured in the medium with 0.2 mg/1 2,4-D (+ D culture). A modified Murashige and Skoog medium [10] was used for the cell culture, in which the concentration of NH4NO3 and KNO3 were at 10.3 mM and 24.7 mM, respectively. Organic addenda were as follows: 3 mg/1 thiamine--HC1, 5 mg/1 nicotinic acid, 0.5 mg/1 pyridoxine-HC1, 100 mg/1 m-inositol, 500 mg/1 casein hydrolysate, 20 g/1 sucrose. All the suspensions were maintained in 300-ml flasks placed on a reciprocal shaker at 26°C under continuous light of 1500 Ix intensity. At the end of culture, the cells were washed with H20 and frozen at -40°C. DNA preparation The DNA was prepared from the crude chromatin fraction. The cells were pulverized in a chilled mortar, then treated in a Potter homogenizer in 0.3 M sucrose, 0.05 M Tris (pH 8.0), 0.001 M MgC12 and 0.01 M 2-mercaptoethanol. The homogenate was subsequently filtered through a triplelayered gauze and Miracloth. The filtrate was centrifuged at 4000 X g for 30 min. The pellet was suspended in 0.3 M sucrose, 0.05 M Tris (pH 8.0)
25 and 0.01 M 2-mercaptoethanol, and centrifuged at 10 000 X g for 10 rain. The pelletwas resuspended in the same medium, layered on a 1.7 M sucrose in 0.05 M Tris (pH 8.0) and then centrifuged at 63 000 × g (av.)for 120 min at 4°C. The resultingpelletwas dissolvedin 0.1 M Tris (pH 7.6),0.05 M NaCI, 0.01 M E D T A , 2% sodium laurylsulfate(SLS) and 25"~I/mI diethylpyrocarbonate to 0.5 M NaCI, the mixture was centrifuged at 8000 X g for 5 min. The s u p e m a t a n t was depmteinized with an equal volume o f chloroform/isoamylalcohol (24: 1), and the nucleic acids were precipitated by the addition of 2 vol. of 95% ethanol. The precipitate was dissolved in 1 × standard sodium citrate buffer (SSC) (0.15 M NaC1, 0.015 M trisodium citrate, pH 7.2), and the solution was incubated with RNase (50 pl/ml, pancreatic) for 90 rain and then Pronase E (50 #g/ml) for 60 min at 37°C. After extraction with chloroform/isoamylalcohol, DNA was finally purified on a CsCl density gradient.
Preparation of radioactive rRNA Carrot cells were cultured with 10 pCi/ml [14C]uridine {The Radiochemical Centre, Amersham, U.K., 57 Ci/mmol) for 50 h. Nucleic acids were extracted by the m e t h o d of Laulhere and Rozier [11]. The final precipitate of nucleic acids was suspended in 3 M sodium acetate (pH 6.0) and centrifuged at 20 000 × g for 10 rain. The supernatant which contains DNA and sRNA was discarded, and the precipitate was dissolved in 0.1 M sodium acetate {pH 6.0) with 1 mM EDTA. This R N A solution was fractionated on a 5--30% sucrose gradient. The 25 S and 18 S RNAs were further purified on sucrose gradients, combined on a equal molecular basis and used for hybridization. The specific activity was 6900 cpm/~g RNA.
Hybridization DNA in 0.1 × SSC was denatured b y the addition of 0.1 vol. of 0.5 M NaOH for 10 rain at room temperature. The solution was neutralized by the addition of 1 M KH2PO4, and the salt concentration was adjusted to 6 × SSC. The denatured DNA was loaded onto membrane filters (Schleicher and Schiill, BA85), which were then washed with 6 × SSC, air-dried and baked at 80°C for 2 h. All the hybridization was carried o u t in 50% formamide at 36°C in 2 × SSC for 16 h [18]. The filters were then washed successively in five changes of 2 × SSC, after which they were incubated with RNase (20 pg/ml, pancreatic) for 40 min at 25°C followed by additional wash in 2 × SSC. The filters were dried and the radioactivity was determined. The DNA content of the filter was routinely monitored by acid hydrolysis. Extraction and determination of nucleic acids The nucleic acids were fractionated by the method of Mizuno and Whiteley [12]. The amounts of R N A and D N A were determined by orcinol [13] and diphenylamine reaction [14], respectively.
26
The DNA content per nucleus of cultured cells was measured by comparative Feulgen photometry, using Allium cepa (33.5 pg per telophase nucleus) as a standard [ 15 ]. RESULTS The developmental sequence of embryogenesis was divided into four stages of the process: globular, heart, t ~ o Snd'plantlet stages. As shown in Table I, embryogenesis was trig~ereti"~%e~ transferring the cells into --D culture. The inoculated cell chuiiL~s~gan %0 acquire the distinctive structure of embryos on day 4:aud~4~he.' number of embryos increased rapidly from day 4 to day i0. Tl~.en~bryosprogressed through development and about 37% of total:embry0s established the formation of torpedo embryos or plantlets within 14 days. On the other hand, the + D culture did not show any sign of embryogenesis throughout the 14-day culture period. Time-course changes in RNA content and in the ratio of RNA:DNA in the cultures were examined to compare the results between cultures with and without 2,4-D. The increase of RNA content in both cultures was apparent during the e x p e r i m ~ t a l period (Fig. 1A). However, during the first 8 days, the rate of RNA increase in --D culture exceeded that of + D culture also after day 8. The RNA:DNA ratios in both cultures rapidly increased in the first 4 days (Fig. 1B). The --D culture remained an elevated level of the ratio from day 4 to day 6, after which time the ratio decreased below the initial value, while the ratio in +D culture declined steeply between days 4 and 10. The maximum ratio in --D culture was markedly higher than the maximum ratio in + D culture. The determination of nuclear DNA content by micro-
TABLE I SOMATIC EMBRYOGENESISIN CARROT CELL SUSPENSIONCULTURES GROWING IN THE ABSENCE OF 2,4-D Day
Total No./ml culture
Embryos Distribution of stages (%) Globular and heart
0 2 4 6 8 10 14
0 0 2 252 678 1086 1016
-
-
100 93.5 72.0 69.7 63.1
Torpedo and plantlet m
0 6.5 28.0 30.3 36.9
27 100 (~
<
®
100 t
10 <( Z E3
IZ
•Z
~
1
50
Z (lC
ne
E 0.1 I
0
4
I
/
I
8 DAY
I
O
12
.
0
.
,
A
4
,
,
,
8 DAY
12
Fig. 1. C h a n g u o f RNA content (A) and o f RNA :DNA ratio (B) in carrot cell suspenlion cultures with (e =) and without (o o) 2,4-D. T h e bars r e p r ~ e n t S.D. (n = 3).
spectrophotometry showed no significant variation in the ploidy level throughout the culture period regardless of the presence of 2,4-D. The rDNA amount was determined in each DNA preparation obtained at the various stages of the cultures. Preliminarily the condition o f hybridization was assessed. RNA at 4 #g/ml in the reaction mixture was required for saturation o f hybridization with 13 #g DNA/filter. Therefore, to ensure saturation, all hybridizations were carried out at 8 #g/ml RNA. Thermal denaturation profiles yielded a Tm of 89°C for the hybrid in 1 X SSC, which implied that precise base-pairing took place between rRNA and DNA. As shown in Table II, the --D culture showed a slight increase in rDNA amount
T A B L E II r D N A A M O U N T S IN C A R R O T C E L L S U S P E N S I O N C U L T U R E S
Thirteen ~g D N A were loaded onto a filter, and 11.78 ± 0.29 ~g of the DNA were remined on the filter after determination of radioactivity. Hybridized values were calculated from the duplicate experiments using separate materials. Day
0 3 6 12
+ 2,4-D
- - 2,4-D
% DNA hybridized
Relative %
% DNA hybridized
Relative %
0.599 0.594 0.622 0.608
100 99 104 102
-0.609 ¢ 0.023 0.673 ± 0.005 0.605 ± 0.013
-102 112 101
+ ± ± ±
0.005 0.002 0.003 0.020
28
on day 6, but the extent of increase was merely 12% higher than the value at the inoculation time. The rDNA amount in --D culture, when the culture period was prolonged to 12 days, decreased down to the same level as the value obtained from inoculum cells. The rDNA amount in the cells of + D culture exhibited no appreciable change through the experimental period.
DISCUSSION It was demonstrated that the composition of nucleic acids in carrot cell suspension cultures considerably changed during their development. Since the ploidy of the cells is constant, the changes in RNA : DNA ratio during the development of the culture reflect changes in RNA content per cell. Furthermore, the differences in RNA content of the cells between --D culture and + D culture may be clarified by making a comparison of RNA : DNA ratios under the two conditions. The --D culture accumulates RNA at a high rate prior to the formation of embryos (the contribution of rRNA was 75--85% of total RNA, unpublished data), while the RNA content of cells decreased in advance of their development (Fig. 1). It has been found that the variations of rRNA gene redundancy between different growth phases of ivy were not responsible for the increase in RNA content associated with the phase changes [9]. On the other hand, the preferential replication of satellite DNA has been indicated using the cultured tissues of carrot explants [16]. In this case, however, no significant differences in the amount and structure of rDNA were found between carrot explants and the calluses,suggesting that the r R N A genes were stable during the process of dedifferentiation in tissue culture [17]. The rapid production of R N A in the vigorous stage of development of embryogenic culture m a y require quantitative change in r R N A genes. As indicated in Table II, the percentage of D N A which hybridized to r R N A showed a slight change during embryogenic culture. However it is stilla matter of controversy whether this temporary change is due to the differential replication of rDNA. As technical problems in making comparative estimates of r D N A amount, Ingle and Sinclair [19] have pointed out the molecular weight of purified D N A , differentialsticking of D N A to membrane filtersand preferentialloss of D N A from filtersduring hybridization, etc. The present experiments could not entirely exclude some of these problems for the following reasons: (I) carrot genome size was very small (1 pg per 2n nucleus) and hence the purification steps of D N A had to be complicated, (2) the formation of globular embryos rapidly reduced the cell volume, which should criticallyinfluence the degree of mechanical shearing of D N A during extraction. Therefore, taking into account these experimental difficulties,it was concluded that the embryogenic culture of carrot cellsdid not associate with the quantitative change in rDNA. These findings also suggest that the production of a large amount of r R N A for occasional requirement m a y not be regulated by the temporary change of r R N A genes.
29 REFERENCES
1 W. Halperin, Am. J. Bot., 53 (1966) 443. 2 K. Muuda, Y. Kikuta and Y. Okazawa, J. Fac. Agric. Hokkaido Univ., 60 (1981) 183. 3 H. Matsumoto, D. Gregor and J. Reinert, Phytochemistry, 14 (1975) 41. 4 T. Fujimura, A. Komamine and H. Matsumoto, Physiol. Plant., 49 (1980) 255. 5 F.C. Steward, M.O. Mapes, A.E. Kent and R.D. Holsten, Science, 143 (1964) 20. 6 A. Siegel, Gene amplification in plants, in: R. Markham, D.R. Davies, D.A. Hopwood and R.W. Home (Eds.), Modification of the Information Content of Plant Cells, North-Holland, Amsterdam, 1975, p. 15. 7 J.N. Timmis and J. Ingle, Eiochem. Genet., 13 (1975) 629. 8 C.A. Cullis and L. Charlton, Plant Sci. Lett., 20 (1981) 218. 9 C. Domoney and J.N. Timmis, J. Exp. Bot., 31 (1980) 1093. 10 T. Murashige and F. Skoog, Physiol. Plant., 15 (1962) 473. 11 J.P. Laulhere and C. Rozier, Plant Sci. Left., 6 (1976) 237. 12 S. Mizuno and H.R. Whiteley, J. Bacteriol., 95 (1968) 1221. 13 W. Mejbaum, Z. Physiol. Chem., 258 (1939) 117. 14 K.W. Giles and A. Myers, Nature, 206 (1965) 93. 15 J. McLeish and N. Sunderland, Exp. Cell Res., 24 (1961) 527. 16 Y. Hue, K. Yakura and S. Tanffuji, Plant and Cell Physiol., 20 (1979) 1461. 17 A. Kato, K. Yakura and S. Tanifuji, Plant and Cell Physiol., 23 (1982) 151. 18 A. Jaworski and L. Key, Plant Physiol., 53 (1974) 366. 19 J. Ingle and J. Sinclair, Nature, 235 (1972) 30.
23
Elsevier Scientific Publishers Ireland Ltd.
EMBRYOGENESIS AND RIBOSOMAL DNA IN CARROT CELL SUSPENSIONS CULTURED IN VITRO
KIYOSHI MASUDA, YOSHIO KIKUTA and YOZO OKAZAWA
Faculty of Agriculture, Department of Botany, Hokkaido University, Sapporo, 060 (Japan) (Received January 31st, 1983) (Revision received July 13th, 1983) (Accepted July 13th, 1983)
SUMMARY
The contents of nucleic acids and rDNA were estimated during the development of carrot cell suspensions cultured under two different conditions. The cells transferred from stock culture to the medium without 2,4-dichlorophenoxyacetic acid (2,4-D) induced the embryogenesis (embryogenic culture~ while the cells inoculated to the medium with 0.2 mg/l 2,4-D did not form any embryos (non-embryogenic culture). The ratio of RNA to DNA of both cultures increased in the early stage of the culture. The rise of the ratio in embryogenic culture was much higher than that in non-embryogenic culture, which showed that embryogenic culture accumulated RNA prior to the formation of embryos. The rDNA amount of non-embryogenic culture remained constant throughout the culture period. Although embryogenic culture showed a slight change in rDNA amount, the differences were at most 12% and the quantitative stability of the rDNA was demonstrated during the development of carrot cell suspension cultures. K e y w o r d s : D a u c u s c a r o t a - - Somatic embryogenesis -- rDNA
INTRODUCTION
It has been well documented that somatic embryogenesis in carrot cell suspension is readily elicited when cells cultured in a medium supplemented with 2,4-D are subsequently transferred to an auxin-free medium [1,2]. It may be expected that changes of cell metabolism in culture would have to occur at an early stage of embryogenic culture. In fact, much attention Abbreviations: 2,4-D, 2,4-dichlorophenoxyacetic acid; SLS, sodium lauryl sulfate; SSC, standard sodium citrate buffer. 0304-4211/84/$03.00 © 1984 Elsevier Scientific Publishers Ireland Ltd. Printed and Published in Ireland
24
has been paid to embryogenesis in relation to a pronounced accumulation of RNA [5] and to a noticeable stimulation in chromatin~iirected RNA synthesis [3,4]. Although it is generally known that plant cells contain a large number of copies of rRNA genes, little information is presently available on the evidence for their differential replication [6]. For instance it has been reported that the changes of rDNA amount of flax plants seem to be closely associated with the phenotypical modification, which is induced by the alteration of nutritional and environmental conditions [7,8]. It may be anticipated, therefore, that the rRNA genes in plants would also be liable to change in advance of their development. In the present study, the question arises whether redifferentiation in plant cell cultures is linked with the quantitative change of rRNA genes. The cell suspension culture of carrot provides a suitable system for evaluation of this problem. In this paper, rDNA amounts in carrot cell suspensions were estimated during their developmental period, and the possibility was explored that RNA accumulation at the early stage of embryogenic culture would be associated with a quantitative variation of rRNA genes. MATERIALS AND METHODS
Culture The suspension cultures of carrot (Daucus carota L. cv. Red Core Chantenay) were established from sterile seedlings and maintained in liquid medium with 0.2 rag/1 2,4-D [2]. The inocula for embryogenic culture were prepared from 13-day-old stock culture. The cells passed through a 177-~m metal screen were collected on a 44-~m screen, washed with fresh medium and inoculated to medium without 2,4-D (--D culture). For non-embryogenic culture, the inocula were subcultured in the medium with 0.2 mg/1 2,4-D (+ D culture). A modified Murashige and Skoog medium [10] was used for the cell culture, in which the concentration of NH4NO3 and KNO3 were at 10.3 mM and 24.7 mM, respectively. Organic addenda were as follows: 3 mg/1 thiamine--HC1, 5 mg/1 nicotinic acid, 0.5 mg/1 pyridoxine-HC1, 100 mg/1 m-inositol, 500 mg/1 casein hydrolysate, 20 g/1 sucrose. All the suspensions were maintained in 300-ml flasks placed on a reciprocal shaker at 26°C under continuous light of 1500 Ix intensity. At the end of culture, the cells were washed with H20 and frozen at -40°C. DNA preparation The DNA was prepared from the crude chromatin fraction. The cells were pulverized in a chilled mortar, then treated in a Potter homogenizer in 0.3 M sucrose, 0.05 M Tris (pH 8.0), 0.001 M MgC12 and 0.01 M 2-mercaptoethanol. The homogenate was subsequently filtered through a triplelayered gauze and Miracloth. The filtrate was centrifuged at 4000 X g for 30 min. The pellet was suspended in 0.3 M sucrose, 0.05 M Tris (pH 8.0)
25 and 0.01 M 2-mercaptoethanol, and centrifuged at 10 000 X g for 10 rain. The pelletwas resuspended in the same medium, layered on a 1.7 M sucrose in 0.05 M Tris (pH 8.0) and then centrifuged at 63 000 × g (av.)for 120 min at 4°C. The resultingpelletwas dissolvedin 0.1 M Tris (pH 7.6),0.05 M NaCI, 0.01 M E D T A , 2% sodium laurylsulfate(SLS) and 25"~I/mI diethylpyrocarbonate to 0.5 M NaCI, the mixture was centrifuged at 8000 X g for 5 min. The s u p e m a t a n t was depmteinized with an equal volume o f chloroform/isoamylalcohol (24: 1), and the nucleic acids were precipitated by the addition of 2 vol. of 95% ethanol. The precipitate was dissolved in 1 × standard sodium citrate buffer (SSC) (0.15 M NaC1, 0.015 M trisodium citrate, pH 7.2), and the solution was incubated with RNase (50 pl/ml, pancreatic) for 90 rain and then Pronase E (50 #g/ml) for 60 min at 37°C. After extraction with chloroform/isoamylalcohol, DNA was finally purified on a CsCl density gradient.
Preparation of radioactive rRNA Carrot cells were cultured with 10 pCi/ml [14C]uridine {The Radiochemical Centre, Amersham, U.K., 57 Ci/mmol) for 50 h. Nucleic acids were extracted by the m e t h o d of Laulhere and Rozier [11]. The final precipitate of nucleic acids was suspended in 3 M sodium acetate (pH 6.0) and centrifuged at 20 000 × g for 10 rain. The supernatant which contains DNA and sRNA was discarded, and the precipitate was dissolved in 0.1 M sodium acetate {pH 6.0) with 1 mM EDTA. This R N A solution was fractionated on a 5--30% sucrose gradient. The 25 S and 18 S RNAs were further purified on sucrose gradients, combined on a equal molecular basis and used for hybridization. The specific activity was 6900 cpm/~g RNA.
Hybridization DNA in 0.1 × SSC was denatured b y the addition of 0.1 vol. of 0.5 M NaOH for 10 rain at room temperature. The solution was neutralized by the addition of 1 M KH2PO4, and the salt concentration was adjusted to 6 × SSC. The denatured DNA was loaded onto membrane filters (Schleicher and Schiill, BA85), which were then washed with 6 × SSC, air-dried and baked at 80°C for 2 h. All the hybridization was carried o u t in 50% formamide at 36°C in 2 × SSC for 16 h [18]. The filters were then washed successively in five changes of 2 × SSC, after which they were incubated with RNase (20 pg/ml, pancreatic) for 40 min at 25°C followed by additional wash in 2 × SSC. The filters were dried and the radioactivity was determined. The DNA content of the filter was routinely monitored by acid hydrolysis. Extraction and determination of nucleic acids The nucleic acids were fractionated by the method of Mizuno and Whiteley [12]. The amounts of R N A and D N A were determined by orcinol [13] and diphenylamine reaction [14], respectively.
26
The DNA content per nucleus of cultured cells was measured by comparative Feulgen photometry, using Allium cepa (33.5 pg per telophase nucleus) as a standard [ 15 ]. RESULTS The developmental sequence of embryogenesis was divided into four stages of the process: globular, heart, t ~ o Snd'plantlet stages. As shown in Table I, embryogenesis was trig~ereti"~%e~ transferring the cells into --D culture. The inoculated cell chuiiL~s~gan %0 acquire the distinctive structure of embryos on day 4:aud~4~he.' number of embryos increased rapidly from day 4 to day i0. Tl~.en~bryosprogressed through development and about 37% of total:embry0s established the formation of torpedo embryos or plantlets within 14 days. On the other hand, the + D culture did not show any sign of embryogenesis throughout the 14-day culture period. Time-course changes in RNA content and in the ratio of RNA:DNA in the cultures were examined to compare the results between cultures with and without 2,4-D. The increase of RNA content in both cultures was apparent during the e x p e r i m ~ t a l period (Fig. 1A). However, during the first 8 days, the rate of RNA increase in --D culture exceeded that of + D culture also after day 8. The RNA:DNA ratios in both cultures rapidly increased in the first 4 days (Fig. 1B). The --D culture remained an elevated level of the ratio from day 4 to day 6, after which time the ratio decreased below the initial value, while the ratio in +D culture declined steeply between days 4 and 10. The maximum ratio in --D culture was markedly higher than the maximum ratio in + D culture. The determination of nuclear DNA content by micro-
TABLE I SOMATIC EMBRYOGENESISIN CARROT CELL SUSPENSIONCULTURES GROWING IN THE ABSENCE OF 2,4-D Day
Total No./ml culture
Embryos Distribution of stages (%) Globular and heart
0 2 4 6 8 10 14
0 0 2 252 678 1086 1016
-
-
100 93.5 72.0 69.7 63.1
Torpedo and plantlet m
0 6.5 28.0 30.3 36.9
27 100 (~
<
®
100 t
10 <( Z E3
IZ
•Z
~
1
50
Z (lC
ne
E 0.1 I
0
4
I
/
I
8 DAY
I
O
12
.
0
.
,
A
4
,
,
,
8 DAY
12
Fig. 1. C h a n g u o f RNA content (A) and o f RNA :DNA ratio (B) in carrot cell suspenlion cultures with (e =) and without (o o) 2,4-D. T h e bars r e p r ~ e n t S.D. (n = 3).
spectrophotometry showed no significant variation in the ploidy level throughout the culture period regardless of the presence of 2,4-D. The rDNA amount was determined in each DNA preparation obtained at the various stages of the cultures. Preliminarily the condition o f hybridization was assessed. RNA at 4 #g/ml in the reaction mixture was required for saturation o f hybridization with 13 #g DNA/filter. Therefore, to ensure saturation, all hybridizations were carried out at 8 #g/ml RNA. Thermal denaturation profiles yielded a Tm of 89°C for the hybrid in 1 X SSC, which implied that precise base-pairing took place between rRNA and DNA. As shown in Table II, the --D culture showed a slight increase in rDNA amount
T A B L E II r D N A A M O U N T S IN C A R R O T C E L L S U S P E N S I O N C U L T U R E S
Thirteen ~g D N A were loaded onto a filter, and 11.78 ± 0.29 ~g of the DNA were remined on the filter after determination of radioactivity. Hybridized values were calculated from the duplicate experiments using separate materials. Day
0 3 6 12
+ 2,4-D
- - 2,4-D
% DNA hybridized
Relative %
% DNA hybridized
Relative %
0.599 0.594 0.622 0.608
100 99 104 102
-0.609 ¢ 0.023 0.673 ± 0.005 0.605 ± 0.013
-102 112 101
+ ± ± ±
0.005 0.002 0.003 0.020
28
on day 6, but the extent of increase was merely 12% higher than the value at the inoculation time. The rDNA amount in --D culture, when the culture period was prolonged to 12 days, decreased down to the same level as the value obtained from inoculum cells. The rDNA amount in the cells of + D culture exhibited no appreciable change through the experimental period.
DISCUSSION It was demonstrated that the composition of nucleic acids in carrot cell suspension cultures considerably changed during their development. Since the ploidy of the cells is constant, the changes in RNA : DNA ratio during the development of the culture reflect changes in RNA content per cell. Furthermore, the differences in RNA content of the cells between --D culture and + D culture may be clarified by making a comparison of RNA : DNA ratios under the two conditions. The --D culture accumulates RNA at a high rate prior to the formation of embryos (the contribution of rRNA was 75--85% of total RNA, unpublished data), while the RNA content of cells decreased in advance of their development (Fig. 1). It has been found that the variations of rRNA gene redundancy between different growth phases of ivy were not responsible for the increase in RNA content associated with the phase changes [9]. On the other hand, the preferential replication of satellite DNA has been indicated using the cultured tissues of carrot explants [16]. In this case, however, no significant differences in the amount and structure of rDNA were found between carrot explants and the calluses,suggesting that the r R N A genes were stable during the process of dedifferentiation in tissue culture [17]. The rapid production of R N A in the vigorous stage of development of embryogenic culture m a y require quantitative change in r R N A genes. As indicated in Table II, the percentage of D N A which hybridized to r R N A showed a slight change during embryogenic culture. However it is stilla matter of controversy whether this temporary change is due to the differential replication of rDNA. As technical problems in making comparative estimates of r D N A amount, Ingle and Sinclair [19] have pointed out the molecular weight of purified D N A , differentialsticking of D N A to membrane filtersand preferentialloss of D N A from filtersduring hybridization, etc. The present experiments could not entirely exclude some of these problems for the following reasons: (I) carrot genome size was very small (1 pg per 2n nucleus) and hence the purification steps of D N A had to be complicated, (2) the formation of globular embryos rapidly reduced the cell volume, which should criticallyinfluence the degree of mechanical shearing of D N A during extraction. Therefore, taking into account these experimental difficulties,it was concluded that the embryogenic culture of carrot cellsdid not associate with the quantitative change in rDNA. These findings also suggest that the production of a large amount of r R N A for occasional requirement m a y not be regulated by the temporary change of r R N A genes.
29 REFERENCES
1 W. Halperin, Am. J. Bot., 53 (1966) 443. 2 K. Muuda, Y. Kikuta and Y. Okazawa, J. Fac. Agric. Hokkaido Univ., 60 (1981) 183. 3 H. Matsumoto, D. Gregor and J. Reinert, Phytochemistry, 14 (1975) 41. 4 T. Fujimura, A. Komamine and H. Matsumoto, Physiol. Plant., 49 (1980) 255. 5 F.C. Steward, M.O. Mapes, A.E. Kent and R.D. Holsten, Science, 143 (1964) 20. 6 A. Siegel, Gene amplification in plants, in: R. Markham, D.R. Davies, D.A. Hopwood and R.W. Home (Eds.), Modification of the Information Content of Plant Cells, North-Holland, Amsterdam, 1975, p. 15. 7 J.N. Timmis and J. Ingle, Eiochem. Genet., 13 (1975) 629. 8 C.A. Cullis and L. Charlton, Plant Sci. Lett., 20 (1981) 218. 9 C. Domoney and J.N. Timmis, J. Exp. Bot., 31 (1980) 1093. 10 T. Murashige and F. Skoog, Physiol. Plant., 15 (1962) 473. 11 J.P. Laulhere and C. Rozier, Plant Sci. Left., 6 (1976) 237. 12 S. Mizuno and H.R. Whiteley, J. Bacteriol., 95 (1968) 1221. 13 W. Mejbaum, Z. Physiol. Chem., 258 (1939) 117. 14 K.W. Giles and A. Myers, Nature, 206 (1965) 93. 15 J. McLeish and N. Sunderland, Exp. Cell Res., 24 (1961) 527. 16 Y. Hue, K. Yakura and S. Tanffuji, Plant and Cell Physiol., 20 (1979) 1461. 17 A. Kato, K. Yakura and S. Tanifuji, Plant and Cell Physiol., 23 (1982) 151. 18 A. Jaworski and L. Key, Plant Physiol., 53 (1974) 366. 19 J. Ingle and J. Sinclair, Nature, 235 (1972) 30.