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Growth and coloration of Tradescantia stamen hairs cultured in vitro
Environmental and Experimental Botany, Vol. 22, No. 2, pp. 255 to 261, 1982 Printed in Great Britain
0098-8472/82/020255-07 $03.00/0 ~ 1982. Pergamon Press Ltd.
SHORT COMMUNICATION
G R O W T H AND C O L O R A T I O N OF T R A D E S C A N T I A STAMEN HAIRS C U L T U R E D I N VITRO* CHIZU NAGASHIMA-ISHII and SADAO ICHIKAWA
Laboratory of Genetics, Department of Regulation Biology, Faculty of Science, Saitama University, Urawa 338, Japan (Received 19 December 1980; in revisedform 5 March 1981; infinal form 19 April 1981) NAGASHIMA-IsHnC. and ICHIKAWAS. Growth and coloration 0fTradescantia stamen hairs cultured in vitro. ENVIRONMSNTALAND EXP~gI~ENTAL BOTANY 22, 255--261, 1982.--Young stamens of Tradescantia clone 02 were cultured in vitro on agar media containing various combinations of three plant hormones, IAA, GA 3 and kinetin. Maximum growth of stamen hairs was observed with medium containing 5.0 mg/1 IAA, 1.0 mg/1 GA 3 and 0.1 mg/1 kinetin. As much as a 10 cell increase was observed in stamen hairs on this medium, but the growth did not equal the normal in vivo growth. The hormonal concentrations of the medium which induced maximum coloration were 0.1 rag/1 IAA, 1.0 mg/1 GA 3 and 0.1 ms/1 kinetin. Higher concentrations of sucrose also seemed effective in promoting coloration. The distribution pattern of colored cells along hairs (more coloration at hair tips) supports the idea that completion of cell division is one of the 'triggers' of coloration. The present study suggests the possibility of developing in vitro culture techniques of Tradescantia stamens. INTRODUCTION
IT Is widely k n o w n that Tradescantia species have features p a r t i c u l a r l y well suited for certain radiobiological studiesJ 4-6) T h e stamen hairs of some Tradescantia clones have been useful in studying i n d u c e d somatic m u t a t i o n frequencies at millirad levels of r a d i a t i o n doses.t 1,2,14) T h e techniques for using the s t a m e n - h a i r system developed over the past d e c a d e from radiobiological studies, have found a p p l i c a t i o n in experiments with known chemical mutagens a n d other environm e n t a l substancesJ 3,a 5) SPARROW and S H A I R E R (12) a n d S P A R R O W et alJX 3) treated Tradescantia stamen hairs with some chemicals in a gaseous form. ICHIKAWA(3) a n d ICHIKAWA a n d TAI~AI-IASm(7) developed a m e t h o d
for a p p l y i n g chemical mutagens as aqueous solutions. W i t h these methods, however, the concentrations of m u t a g e n s which p e n e t r a t e d into the tissue r e m a i n e d uncertain, a n d thus, it was desirable to develop a new m e t h o d with which y o u n g stamen hairs could be directly exposed to chemical mutagens. T h e present p a p e r reports an a t t e m p t to develop an in vitro culture technique o f Tradescantia stamens a p p l i c a b l e for chemical m u t a g e n testing.
MATERIAL A N D M E T H O D S
T h e m a t e r i a l used in the present study was Tradescantia clone 02, originally supplied in 1974 by the late Dr. A. H. SPARROW of Brookhaven
*Research partly carried out at the Laboratory of Genetics, Faculty of Agriculture, Kyoto University. Supported in part by a Grant-in-Aid for Scientific Research (No. 358071) from Ministry of Education, Japan. 255
256
C. NAGASHIMA-ISHII and S. ICHIKAWA
National Laboratory, U.S.A. This clone is a diploid hybrid (2n = 12) and is heterozygous for flower color (blue/pink, the blue being dominant). T h e origin of this clone is not clear, ~8) but the characteristics of the clone are described elsewhere.(6,s,9) Flower buds of 2 4 m m in length were excised from young inflorescences of potted plants grown in an air-conditioned greenhouse (18-23°C; 17 hr daylength). Excised buds were dipped into a 10% solution of sodium hypochlorite to prevent microbial contamination then washed with sterile water three times. After removing sepals and petals and excising one out of six stamens (one antipetalous stamen, see below) of each bud under a stereoscope, the remaining part (composed of five stamens plus ovary) was planted on one of the agar media described below. These young stamens and their hairs were still colorless at the time of the planting. In most cases, 30 buds were used for each medium. WHITE'S basal m e d i u m ~16) w i t h o u t C u S O 4. H 2 0 and M o O 3 was used. Eleven different combinations of indole-3-acetic acid (IAA), gibberellic acid (GA3) and kinefin were
added to the basal medium in the first experiment (Table 1), and 3 out of the 11 combinations were re-examined in the second experiment. Also added were 0.5% agar and 4 % sucrose, except for one medium to which 8% sucrose was added (see Table 1 footnote). T h e excised stamen was used for determining the extent of hair growth in each bud at the time of planting. T h e n u m b e r of hair cells was counted for the basal six hairs of each excised stamen, and the data collected were used for comparison with data taken after culturing. After culturing for 10 days at 21, 23 or 26°C (see Table 1) under continuous light of about 4000 lx, all the stamens were harvested and the cell numbers of the six basal hairs of each stamen were counted to determine hair growth during culture. Hair-cell coloration (from colorless to blue) was also recorded. RESULTS
Experiment l The growth of hairs (increase in cell n u m b e r per hair) which occurred during the 10 day
Table 1. Concentrations of IAA, GA 3 and kinetin supplemented, experimentalperiods, and temperature Medium no.
Experiment 1 1 2 3 4 5 6 7 8 9 10 11 Experiment 2 R1 R3 R5 MI* 0
Concentration (mg/1) IAA GA 3 Kinetin
Experimental period
Cultured at (°C)
0.1 1.0 5.0 0.1 0.1 0.1 0 0 0.1 0.1 0.1
1.0 1.0 1.0 1.0 5.0 5.0 1.0 5.0 5.0 5.0 10.0
0.1 0.1 0.1 0.05 0.05 0.1 0.05 0.1 0.2 0.5 0.5
March March March August August August November November November November November
23 4-1 23 + 1 23 4- 1 26 4- 1 26 4- 1 26+ 1 21 4- 1 21 4- 1 21 4- 1 21 4- 1 21 4- 1
0.1 5.0 0.1 0.1 0
1.0 1.0 5.0 1.0 0
0.1 0.1 0.05 0.1 0
January January January January January
21 4-1 21 4-1 21 4-1 21 4-1 21 4-1
*Contained 8% sucrose. Other media contained 4% sucrose.
T R A D E S C A N T I A STAMEN HAIRS C U L T U R E D I N VITRO
Table 2. The growth (increase in cell number) of three classes of stamens* after 10 day culturing on 11 different media containing various concentrations ofIAA, GA 3 and kinetin (see Table l for the hormone concentrations) Medium no. 1
2
3
4
5
6
7
8
9
10
11
Class of stamens*
No. of buds
I
0
II III I II III I II III I II III I II III I II III I II Ill I II III I II III I II III I II III
3 7 5 8 6 6 5 10 3 8 11 10 15 3 1 6 11 7 15 6 12 7 7 20 7 1 22 5 2 8 12 4
Cell number/hair ( ± SD) Before After culture culture Growth 13.2±0.6 18.2±1.2 8.2±1.3 13.0±0.9 17.9±1.2 8.6±1.1 12.4±1.5 17.0±1.1 7.6±1.5 12.6±1.2 17.1±1.6 7.8±1.5 12.5±1.5 17.0±0.9 9.8 13.4±1.0 17.6±1.5 7.6±2.0 12.8±1.3 16.8±1.2 6.8±2.0 11.3±1.3 17.3±1.5 6.6±2.1 10.9±0.7 15.3 7.3±1.7 12.3±1.7 17.3±1.3 8.4±1.6 12.1±0.8 16.5±0.8
15.3±1.6 20.9±1.8 14.3±0.8 17.5±1.9 20.0±1.7 18.6±2.6 17.8±2.1 18.2±2.4 13.3±0.6 16.0±2.5 19.1±1.6 10.7±2.2 15.4±1.8 17.7±1.2 10.3 17.2±1.1 19.7±1.4 10.0±1.8 14.8±1.6 17.0±1.4 9.2±2.2 12.9±1.7 18.6±2.0 10.1±2.6 14.3±1.8 19.6 t1.1±2.2 13.8±0.5 17.7±1.3 10.8±2.2 14.4±1.4 17.7±0.7
2.1±1.3 2.7±1.4~ 6.0±1.3§ 4.6±1.5§ 2.1±l.Ot 10.0±2.8§ 5.4±3.1§ 1.3±3.0 5.7±2.0§ 3.5±1.9§ 2.0±1.8§ 2.9±1.9§ 2.9±1.8§ 0.6±0.6 0.5 3.8±1.3§ 2.2±1.5§ 2.3±2.5 2.0±1.4§ 0.3±1.2 2.4±1.2~ 1.6±1.3 1.4±1.4 3.5±1.5§ 3.4±1.6§ 4.3 3.8±1.1§ 1.5±1.3 0.4±0.0 2.4±1.6t 2.3±1.6§ 1.2±0.7
*Average cell number per hair examined before culture was less than 10.0 (I), between 10.0 and 14.9 (II), and between 15.0 and 19.9 (IIl). ~'Significant at 5% level. ~:Significant at 2% level. §Significant at 1% level.
257
258
C. NAGASHIMA-ISHII and S. ICHIKAWA
culture on each of 11 media used in Experiment 1 (see Table 1) is presented in Table 2. To analyze the extent of the hair growth, the buds cultured were classified into three classes according to the average n u m b e r of cells per hair in the stamen examined before culture; namely, average cell n u m b e r per hair was less than 10.0 (Class I), between 10.0 and 14.9 (Class II), and between 15.0 and 19.9 (Class I I I ) (see Table 2). Statistically highly significant or at least some significant increases in cell n u m b e r occurred with all the 11 media examined. For class I stamens, which should potentially have the greatest growing ability of hairs, the greatest growth was observed with medium 3, followed by media 2 and 4. Significant increases in cell n u m b e r were also observed on other media, except for medium 7 (no or little data were available for media 1 and 6). For class I I stamens, medium 3 also produced the greatest growth, being followed by media 2, 6, 4 and 9. Significant growth was also observed with media 5, 7 and 11 but not with media 1, 8 and 10. For class I I I stamens, which should have much less potential ability of further hair growth, no clear conclusion could be made. For elucidating the interactions of hormones, several comparisons of hair growth were made between different media. The effect of different amounts of I A A with constant amounts of G A 3 and kinetin, determined by comparing media 13; 4 and 7; or 6 and 8 (see Table 1), indicated that growth of stamen hairs was greater with higher concentrations of I A A (see Table 2). Likewise,
comparisons between media 4 and 5, and 10 and 11, indicated that growth of stamen hairs appeared to be greatest with the lowest (1.0mg/1) concentration of G A 3 when the concentration of I A A was 0.1 mg/1 and that of kinetin was 0.05 or 0.5mg/1 (see Tables 1 and 2). Concerning the effect of kinetin, no clear information was obtained. T h e frequencies of coloration which occurred in the stamen hairs after culture are shown in Table 3. Coloration occurred only with four media (1 3 and 9), being most frequent with medium 1. A lower I A A concentration seemed to promote hair coloration. No essential differences in the distributions of colored cells along stamen hairs were found for media 1 3. T h e subterminal cells colored most frequently, the terminal cells next frequently, and the frequency decreased steadily from the third through the tenth cells from the hair tip (see Fig. 1). lO0
I
I
I
80
I
I
I
I
I
o
o
Experiment 1
~-
A
Experiment 2
I
I
Average
....... 60
S
40
I
"" i)
Table 3. Frequency of coloration occurred in the stamen hairs after 10 day culturing onfour different media (see Table 1for the hormone concentrations) Medium no.*
Number of colored stamen hairs per flowert Class I Class II Class I I I
1
2 3 9
4.4 5.5 0
21.0 10.9 2.2 0
31.9 8.7 1.3 2.0
*No coloration occurred on other media (media 4-8, 10 and 11). 1"'Flower' means a cluster of five stamens plus ovary cultured. All the five stamens did not always survive.
20 ¸
o]
I
I
l
" 1 ~ ~ ~ l ~ l ~
1
2
3
4
5
6
7
8
9
IO
ORDER OF CELLS FROM HAIR TIP
FIo. 1. Distribution patterns of colored cells in the terminal 10 cells of the stamen hairs after culturing for 10 days. Pooled data from Experiment 1 (on media 1, 2 and 3) and those from Experiment 2 (on media R 1, R3, R5 and M1) are compared.
TRADESCANTIA
STAMEN HAIRS CULTURED I N VITRO
259
Table 4. The growth (increase in cell number) of three classes of stamens* after 10 day culturing onfive different media (see Table 1 for the hormone concentrations) Medium no.
Class of stamens*
No. of buds
R1
I II III I II III I II III I II III I II III
11 14 2 12 9 8 16 12 2 9 15 3 7 10 0
R3
R5
MI
0
Cell number/hair ( ± SD) Before After culture culture Growth 7.9±1.1 12.6±1.5 15.9±0.2 7.2±1.8 12.5±1.3 17.4±1.4 8.2±1.2 12.0±1.1 17.9±0.7 8.3±1.4 12.0±1.4 17.4±0.8 8.5±1.2 11.5±1.3
12.6±1.8 17.6±1.6 19.4±0.6 10.8±1.8 16.3±2.5 19.3±1.4 14.0±1.6 16.0±0.8 20.2±0.4 13.6±1.5 15.6±2.5 19.2±1.5 tl.0±1.5 13.6±2.4
4.7±1.1~ 5.0±1.5~ 3.6±0.5t 3.6±1.1~ 3.8±1.4~ 1.9±0.8t 5.8±1.5~ 4.0±0.9~ 2.3±0.3 5.3±2.5~ 3.6±2.0~ 1.9±0.7 2.5±2.1~ 2.0±1.6~
*Average cell number per hair examined before culture was less than 10.0 (I), between 10.0 and 14.9 (II), and between 15.0 and 19.9 (III). ]'Significant at 5% level. ~Significant at 1% level.
Experiment 2 Media l, 3 and 5 used in Experiment 1 were reexamined in Experiment 2, and were designated as media R I , R3 and R5, respectively. Also, a medium with the addition of only agar and sucrose (no plant hormone added) to the basal medium was examined (medium 0). Furthermore, a modified m e d i u m (medium M1) containing 8% instead of 4 % sucrose was included to test the influence of sucrose concentration (see Table 1). Statistically significant increases in cell n u m b e r were observed with all the five media (Table 4), but better growth was observed on media R 1, R5 and M1 rather than on medium R3 which provided the m a x i m u m growth in Experiment 1 (medium 3). Some significant growth of stamen hairs was observed on medium 0, although the increases in cell n u m b e r determined were small (see Table 4). T h e data for hair-cell coloration are shown in Table 5. M e d i u m M1 resulted in the best color-
at±on, far exceeding other media. No coloration was observed on medium 0, suggesting that hormones are required for coloration. T h e distributions of colored cells in the hairs of the stamens cultured on media R1, R3, R5 and M1 were similar to each other, and the distribution pattern
Table 5. Frequency of coloration occurred in the stamen hairs after 10 day culturing onfive different media (see Table l for the hormone concentrations) Medium no. R1 R3 R5 M1 0
Number of colored stamen hairs per flower* Class I Class II Class I I I O.5 3.6 7.6 2.6 0
9.6 13.7 14.2 21.8 0
23.5 9.9 24.0 80.7
*'Flower' means a cluster of five stamens plus ovary cultured. All the five stamens did not always survive.
260
C. N A G A S H I M A - I S H I I and S. I C H I K A W A
is d r a w n in Fig. 1 together with that observed in E x p e r i m e n t 1. T h e results are similar in b o t h experiments.
DISCUSSION
Culture of Tradescantia stamen hairs was first tried by PARCHMANt l ° ' l l ) using T. paludosa, to study h o r m o n a l effects on s t a m e n hair cells. H e observed m a x i m u m growth on m e d i u m containing 10-VM I A A a n d 1 0 - 6 M kinetin. T h e conditions allowed, however, division of s t a m e n hair cells to occur only onceJ TM I n the present study, an increase of as m a n y as 10 cells p e r hair was observed (Table 2). Cell division in Tradescantia s t a m e n hairs is k n o w n to occur mostly at the t e r m i n a l two cells of each hair. ~4~T h e 10 cell increase per hair means that 10 cell divisions on the average occurred p e r hair d u r i n g the culture on the most effective m e d i u m so far examined. T h e difference between PARCHMAN'S a n d our results seems to be due to different culture methods. T h e culture of b a r e stamens resulted in no a p p a r e n t g r o w t h in the p r e l i m i n a r y e x p e r i m e n t c o n d u c t e d before this study. C u l t u r i n g together with other floral organs (e.g. ovary and receptacle) a p p e a r s necessary for successful stamen culture. T h e r e has been no previous report on the coloration of cultured stamen hairs. Results from the present study suggest that coloration m a y be d e t e r m i n e d by h o r m o n a l combinations. Coloration of stamen hairs a n d frequency of colored hairs were most p r o m i n e n t on the m e d i u m which c o n t a i n e d 0.1 mg/1 I A A , 1.0mg/1 G A 3 a n d 0.1mg/1 kinetin (see Tables 1, 3 a n d 5). Sucrose concentrations higher than 4 % also seemed necessary, since the highest frequency of coloration occurred on m e d i u m M1 (see T a b l e s 1 a n d 5). T h e coloration did not occur at r a n d o m . I t was observed that all hairs of a stamen were colored while all hairs of other stamens of the same b u d r e m a i n e d colorless. T h u s s t a m e n - h a i r coloration seemed to be an all-or-nothing event. I n the n o r m a l in vivo flowers of this clone, s t a m e n - h a i r coloration is initiated 5 or 6 days before flowering t6) after c o m p l e t i n g r e p e a t e d divisions of the terminal cell of each hair. T h e completion of the
division is thus t h o u g h t to be a likely 'trigger' of coloration. Distribution patterns of colored cells (from the terminal-cell position to the tenth-cell position from the hair tip) as shown in Fig. 1 indicate that coloration starts at the s u b t e r m i n a l or terminal cells of hairs a n d proceeds to the basal portion. T h e postulate that c o m p l e t i o n of cell division is a 'trigger' of coloration does not c o n t r a d i c t these results, because the terminal a n d s u b t e r m i n a l cells directly receive information of division completion due to the n a t u r e in which cells increase in n u m b e r in stamen hairsJ 4) T h e results of the present study suggest the possibility of developing a successful in vitro culture technique of Tradescantia stamens. Such a technique might be useful a n d inexpensive for routine testing a n d screening of potential chemical and e n v i r o n m e n t a l mutagens.
Acknowledgement--We wish to express our sincere thanks to Prof. K. TSUNEWAKIof Kyoto University for his kindly guidance and encouragement extended to one of us (CNI) during the course of the present study.
REFERENCES 1. ICHIKAWA S. (1971) Somatic m u t a t i o n r a t e at low
levels of chronic gamma-ray exposures in Tradescantia stamen hairs. Jlap. J. Genet. 46, 371381. 2. IGItlKAWA S. (1972) Somatic mutation rate in Tradescantia stamen hairs at low radiation levels: Finding of low doubling doses of mutations, flap. J. Genet. 47, 411-421. 3. IeHIRAWA S. (1976) Tradescantia stamen hairs: A botanical mutagenicity testing system with a high sensitivity. Pages 52-56 in Laboratory manual for
East-Asian workshop on mutagenicity testing of chemicals, National Institute of Genetics, Misima. 4. ICHIKAWA S. and SPARROW A. H. (1967) Radiation-induced loss of reproductive integrity in the stamen hairs of polyploid series of Tradescantia species. Radiat. Bot. 7, 429~41. 5. ICHIKAWAS. and SPARROWA. H. (1968) The use of induced somatic mutations to study cell division rates in irradiated stamen hairs of Tradescantia virginiana L. Jlap. o7 Genet. 43, 57 63. 6. IcHn~AWAS., SPARROWA. H. and THOMPSONK. H. (1969) Morphologically abnormal cells, somatic mutations and loss of reproductive integrity in
TRADESCANTIA
7.
8.
9.
10.
11.
12.
STAMEN HAIRS CULTURED I N VITRO
irradiated Tradescantia stamen hairs. Radiat. Bot. 9, 195-211. IGHIKAWAS. and TAKAHASHIC. S. (1978) Somatic mutations in Tradescantia stamen hairs exposed to ethyl methanesulfonate. Envir. Expl. Bot. 18, 19-25. MERICLE L. W. and MERICLE R. P. (1967) Genetic nature of somatic mutations for flower color in Tradescantia, clone 02. Radiat. Bot. 7, 449464. NAYAR G. G. and SPARROW A. H. (1967) Radiation-induced somatic mutations and loss of reproductive integrity in Tradescantia stamen hairs. Radiat. Bot. 7, 257-267. PARCHMAN L. G. (1962) The effect of certain growth substances upon Tradescantia paludosa stamen hair cells cultured in vitro. Master's Thesis, Emory University. PARCHMANL. G. (1964) The morphogenesis of the stamen hairs of Tradescantia paludosa. Doctor's Thesis, Emory University. SPARROWA. H. and SCHAIRERL. A. (1974) The effects of chemical mutagens (EMS, DBE) and
13.
14.
15.
16.
261
specific air pollutants (O3, SOz, NO2, N/O) on somatic mutation rate in Tradescantia. Text of talk at Symp. on The potential genetic effects of environmental pollutants on man, Moscow, U.S.S.R. Feb. 1974. SPARROWA. H., SCHAIRERL. A. and VILLALOBOSPIETRINI R. (1974) Comparison of somatic mutation rates induced in Tradescantia by chemical and physical mutagens. Murat. Res. 26, 265 276. SPARROW A. H., UNDERBRINK A. G. and RossI H. H. (1972) Mutations induced in Tradescantia by small doses of X-rays and neutrons: Analysis of dose-response curve. Science N. T. 176, 916-918. UNDERBRINKA. G., SCHAIRERL. A. and SPARROW A. H. (1973) Tradescantia stamen hairs: A radiobiological test system applicable to chemical nmtagenesis. Pages 171-207 in A. HOLLAENDER, ed. Chemical mutagens: Principles and methods for their detection, vol. 3, Plenum Press, New York. WHITER. P. (1963) The cultivation of animal andplant cells. Ronald Press, New York.
0098-8472/82/020255-07 $03.00/0 ~ 1982. Pergamon Press Ltd.
SHORT COMMUNICATION
G R O W T H AND C O L O R A T I O N OF T R A D E S C A N T I A STAMEN HAIRS C U L T U R E D I N VITRO* CHIZU NAGASHIMA-ISHII and SADAO ICHIKAWA
Laboratory of Genetics, Department of Regulation Biology, Faculty of Science, Saitama University, Urawa 338, Japan (Received 19 December 1980; in revisedform 5 March 1981; infinal form 19 April 1981) NAGASHIMA-IsHnC. and ICHIKAWAS. Growth and coloration 0fTradescantia stamen hairs cultured in vitro. ENVIRONMSNTALAND EXP~gI~ENTAL BOTANY 22, 255--261, 1982.--Young stamens of Tradescantia clone 02 were cultured in vitro on agar media containing various combinations of three plant hormones, IAA, GA 3 and kinetin. Maximum growth of stamen hairs was observed with medium containing 5.0 mg/1 IAA, 1.0 mg/1 GA 3 and 0.1 mg/1 kinetin. As much as a 10 cell increase was observed in stamen hairs on this medium, but the growth did not equal the normal in vivo growth. The hormonal concentrations of the medium which induced maximum coloration were 0.1 rag/1 IAA, 1.0 mg/1 GA 3 and 0.1 ms/1 kinetin. Higher concentrations of sucrose also seemed effective in promoting coloration. The distribution pattern of colored cells along hairs (more coloration at hair tips) supports the idea that completion of cell division is one of the 'triggers' of coloration. The present study suggests the possibility of developing in vitro culture techniques of Tradescantia stamens. INTRODUCTION
IT Is widely k n o w n that Tradescantia species have features p a r t i c u l a r l y well suited for certain radiobiological studiesJ 4-6) T h e stamen hairs of some Tradescantia clones have been useful in studying i n d u c e d somatic m u t a t i o n frequencies at millirad levels of r a d i a t i o n doses.t 1,2,14) T h e techniques for using the s t a m e n - h a i r system developed over the past d e c a d e from radiobiological studies, have found a p p l i c a t i o n in experiments with known chemical mutagens a n d other environm e n t a l substancesJ 3,a 5) SPARROW and S H A I R E R (12) a n d S P A R R O W et alJX 3) treated Tradescantia stamen hairs with some chemicals in a gaseous form. ICHIKAWA(3) a n d ICHIKAWA a n d TAI~AI-IASm(7) developed a m e t h o d
for a p p l y i n g chemical mutagens as aqueous solutions. W i t h these methods, however, the concentrations of m u t a g e n s which p e n e t r a t e d into the tissue r e m a i n e d uncertain, a n d thus, it was desirable to develop a new m e t h o d with which y o u n g stamen hairs could be directly exposed to chemical mutagens. T h e present p a p e r reports an a t t e m p t to develop an in vitro culture technique o f Tradescantia stamens a p p l i c a b l e for chemical m u t a g e n testing.
MATERIAL A N D M E T H O D S
T h e m a t e r i a l used in the present study was Tradescantia clone 02, originally supplied in 1974 by the late Dr. A. H. SPARROW of Brookhaven
*Research partly carried out at the Laboratory of Genetics, Faculty of Agriculture, Kyoto University. Supported in part by a Grant-in-Aid for Scientific Research (No. 358071) from Ministry of Education, Japan. 255
256
C. NAGASHIMA-ISHII and S. ICHIKAWA
National Laboratory, U.S.A. This clone is a diploid hybrid (2n = 12) and is heterozygous for flower color (blue/pink, the blue being dominant). T h e origin of this clone is not clear, ~8) but the characteristics of the clone are described elsewhere.(6,s,9) Flower buds of 2 4 m m in length were excised from young inflorescences of potted plants grown in an air-conditioned greenhouse (18-23°C; 17 hr daylength). Excised buds were dipped into a 10% solution of sodium hypochlorite to prevent microbial contamination then washed with sterile water three times. After removing sepals and petals and excising one out of six stamens (one antipetalous stamen, see below) of each bud under a stereoscope, the remaining part (composed of five stamens plus ovary) was planted on one of the agar media described below. These young stamens and their hairs were still colorless at the time of the planting. In most cases, 30 buds were used for each medium. WHITE'S basal m e d i u m ~16) w i t h o u t C u S O 4. H 2 0 and M o O 3 was used. Eleven different combinations of indole-3-acetic acid (IAA), gibberellic acid (GA3) and kinefin were
added to the basal medium in the first experiment (Table 1), and 3 out of the 11 combinations were re-examined in the second experiment. Also added were 0.5% agar and 4 % sucrose, except for one medium to which 8% sucrose was added (see Table 1 footnote). T h e excised stamen was used for determining the extent of hair growth in each bud at the time of planting. T h e n u m b e r of hair cells was counted for the basal six hairs of each excised stamen, and the data collected were used for comparison with data taken after culturing. After culturing for 10 days at 21, 23 or 26°C (see Table 1) under continuous light of about 4000 lx, all the stamens were harvested and the cell numbers of the six basal hairs of each stamen were counted to determine hair growth during culture. Hair-cell coloration (from colorless to blue) was also recorded. RESULTS
Experiment l The growth of hairs (increase in cell n u m b e r per hair) which occurred during the 10 day
Table 1. Concentrations of IAA, GA 3 and kinetin supplemented, experimentalperiods, and temperature Medium no.
Experiment 1 1 2 3 4 5 6 7 8 9 10 11 Experiment 2 R1 R3 R5 MI* 0
Concentration (mg/1) IAA GA 3 Kinetin
Experimental period
Cultured at (°C)
0.1 1.0 5.0 0.1 0.1 0.1 0 0 0.1 0.1 0.1
1.0 1.0 1.0 1.0 5.0 5.0 1.0 5.0 5.0 5.0 10.0
0.1 0.1 0.1 0.05 0.05 0.1 0.05 0.1 0.2 0.5 0.5
March March March August August August November November November November November
23 4-1 23 + 1 23 4- 1 26 4- 1 26 4- 1 26+ 1 21 4- 1 21 4- 1 21 4- 1 21 4- 1 21 4- 1
0.1 5.0 0.1 0.1 0
1.0 1.0 5.0 1.0 0
0.1 0.1 0.05 0.1 0
January January January January January
21 4-1 21 4-1 21 4-1 21 4-1 21 4-1
*Contained 8% sucrose. Other media contained 4% sucrose.
T R A D E S C A N T I A STAMEN HAIRS C U L T U R E D I N VITRO
Table 2. The growth (increase in cell number) of three classes of stamens* after 10 day culturing on 11 different media containing various concentrations ofIAA, GA 3 and kinetin (see Table l for the hormone concentrations) Medium no. 1
2
3
4
5
6
7
8
9
10
11
Class of stamens*
No. of buds
I
0
II III I II III I II III I II III I II III I II III I II Ill I II III I II III I II III I II III
3 7 5 8 6 6 5 10 3 8 11 10 15 3 1 6 11 7 15 6 12 7 7 20 7 1 22 5 2 8 12 4
Cell number/hair ( ± SD) Before After culture culture Growth 13.2±0.6 18.2±1.2 8.2±1.3 13.0±0.9 17.9±1.2 8.6±1.1 12.4±1.5 17.0±1.1 7.6±1.5 12.6±1.2 17.1±1.6 7.8±1.5 12.5±1.5 17.0±0.9 9.8 13.4±1.0 17.6±1.5 7.6±2.0 12.8±1.3 16.8±1.2 6.8±2.0 11.3±1.3 17.3±1.5 6.6±2.1 10.9±0.7 15.3 7.3±1.7 12.3±1.7 17.3±1.3 8.4±1.6 12.1±0.8 16.5±0.8
15.3±1.6 20.9±1.8 14.3±0.8 17.5±1.9 20.0±1.7 18.6±2.6 17.8±2.1 18.2±2.4 13.3±0.6 16.0±2.5 19.1±1.6 10.7±2.2 15.4±1.8 17.7±1.2 10.3 17.2±1.1 19.7±1.4 10.0±1.8 14.8±1.6 17.0±1.4 9.2±2.2 12.9±1.7 18.6±2.0 10.1±2.6 14.3±1.8 19.6 t1.1±2.2 13.8±0.5 17.7±1.3 10.8±2.2 14.4±1.4 17.7±0.7
2.1±1.3 2.7±1.4~ 6.0±1.3§ 4.6±1.5§ 2.1±l.Ot 10.0±2.8§ 5.4±3.1§ 1.3±3.0 5.7±2.0§ 3.5±1.9§ 2.0±1.8§ 2.9±1.9§ 2.9±1.8§ 0.6±0.6 0.5 3.8±1.3§ 2.2±1.5§ 2.3±2.5 2.0±1.4§ 0.3±1.2 2.4±1.2~ 1.6±1.3 1.4±1.4 3.5±1.5§ 3.4±1.6§ 4.3 3.8±1.1§ 1.5±1.3 0.4±0.0 2.4±1.6t 2.3±1.6§ 1.2±0.7
*Average cell number per hair examined before culture was less than 10.0 (I), between 10.0 and 14.9 (II), and between 15.0 and 19.9 (IIl). ~'Significant at 5% level. ~:Significant at 2% level. §Significant at 1% level.
257
258
C. NAGASHIMA-ISHII and S. ICHIKAWA
culture on each of 11 media used in Experiment 1 (see Table 1) is presented in Table 2. To analyze the extent of the hair growth, the buds cultured were classified into three classes according to the average n u m b e r of cells per hair in the stamen examined before culture; namely, average cell n u m b e r per hair was less than 10.0 (Class I), between 10.0 and 14.9 (Class II), and between 15.0 and 19.9 (Class I I I ) (see Table 2). Statistically highly significant or at least some significant increases in cell n u m b e r occurred with all the 11 media examined. For class I stamens, which should potentially have the greatest growing ability of hairs, the greatest growth was observed with medium 3, followed by media 2 and 4. Significant increases in cell n u m b e r were also observed on other media, except for medium 7 (no or little data were available for media 1 and 6). For class I I stamens, medium 3 also produced the greatest growth, being followed by media 2, 6, 4 and 9. Significant growth was also observed with media 5, 7 and 11 but not with media 1, 8 and 10. For class I I I stamens, which should have much less potential ability of further hair growth, no clear conclusion could be made. For elucidating the interactions of hormones, several comparisons of hair growth were made between different media. The effect of different amounts of I A A with constant amounts of G A 3 and kinetin, determined by comparing media 13; 4 and 7; or 6 and 8 (see Table 1), indicated that growth of stamen hairs was greater with higher concentrations of I A A (see Table 2). Likewise,
comparisons between media 4 and 5, and 10 and 11, indicated that growth of stamen hairs appeared to be greatest with the lowest (1.0mg/1) concentration of G A 3 when the concentration of I A A was 0.1 mg/1 and that of kinetin was 0.05 or 0.5mg/1 (see Tables 1 and 2). Concerning the effect of kinetin, no clear information was obtained. T h e frequencies of coloration which occurred in the stamen hairs after culture are shown in Table 3. Coloration occurred only with four media (1 3 and 9), being most frequent with medium 1. A lower I A A concentration seemed to promote hair coloration. No essential differences in the distributions of colored cells along stamen hairs were found for media 1 3. T h e subterminal cells colored most frequently, the terminal cells next frequently, and the frequency decreased steadily from the third through the tenth cells from the hair tip (see Fig. 1). lO0
I
I
I
80
I
I
I
I
I
o
o
Experiment 1
~-
A
Experiment 2
I
I
Average
....... 60
S
40
I
"" i)
Table 3. Frequency of coloration occurred in the stamen hairs after 10 day culturing onfour different media (see Table 1for the hormone concentrations) Medium no.*
Number of colored stamen hairs per flowert Class I Class II Class I I I
1
2 3 9
4.4 5.5 0
21.0 10.9 2.2 0
31.9 8.7 1.3 2.0
*No coloration occurred on other media (media 4-8, 10 and 11). 1"'Flower' means a cluster of five stamens plus ovary cultured. All the five stamens did not always survive.
20 ¸
o]
I
I
l
" 1 ~ ~ ~ l ~ l ~
1
2
3
4
5
6
7
8
9
IO
ORDER OF CELLS FROM HAIR TIP
FIo. 1. Distribution patterns of colored cells in the terminal 10 cells of the stamen hairs after culturing for 10 days. Pooled data from Experiment 1 (on media 1, 2 and 3) and those from Experiment 2 (on media R 1, R3, R5 and M1) are compared.
TRADESCANTIA
STAMEN HAIRS CULTURED I N VITRO
259
Table 4. The growth (increase in cell number) of three classes of stamens* after 10 day culturing onfive different media (see Table 1 for the hormone concentrations) Medium no.
Class of stamens*
No. of buds
R1
I II III I II III I II III I II III I II III
11 14 2 12 9 8 16 12 2 9 15 3 7 10 0
R3
R5
MI
0
Cell number/hair ( ± SD) Before After culture culture Growth 7.9±1.1 12.6±1.5 15.9±0.2 7.2±1.8 12.5±1.3 17.4±1.4 8.2±1.2 12.0±1.1 17.9±0.7 8.3±1.4 12.0±1.4 17.4±0.8 8.5±1.2 11.5±1.3
12.6±1.8 17.6±1.6 19.4±0.6 10.8±1.8 16.3±2.5 19.3±1.4 14.0±1.6 16.0±0.8 20.2±0.4 13.6±1.5 15.6±2.5 19.2±1.5 tl.0±1.5 13.6±2.4
4.7±1.1~ 5.0±1.5~ 3.6±0.5t 3.6±1.1~ 3.8±1.4~ 1.9±0.8t 5.8±1.5~ 4.0±0.9~ 2.3±0.3 5.3±2.5~ 3.6±2.0~ 1.9±0.7 2.5±2.1~ 2.0±1.6~
*Average cell number per hair examined before culture was less than 10.0 (I), between 10.0 and 14.9 (II), and between 15.0 and 19.9 (III). ]'Significant at 5% level. ~Significant at 1% level.
Experiment 2 Media l, 3 and 5 used in Experiment 1 were reexamined in Experiment 2, and were designated as media R I , R3 and R5, respectively. Also, a medium with the addition of only agar and sucrose (no plant hormone added) to the basal medium was examined (medium 0). Furthermore, a modified m e d i u m (medium M1) containing 8% instead of 4 % sucrose was included to test the influence of sucrose concentration (see Table 1). Statistically significant increases in cell n u m b e r were observed with all the five media (Table 4), but better growth was observed on media R 1, R5 and M1 rather than on medium R3 which provided the m a x i m u m growth in Experiment 1 (medium 3). Some significant growth of stamen hairs was observed on medium 0, although the increases in cell n u m b e r determined were small (see Table 4). T h e data for hair-cell coloration are shown in Table 5. M e d i u m M1 resulted in the best color-
at±on, far exceeding other media. No coloration was observed on medium 0, suggesting that hormones are required for coloration. T h e distributions of colored cells in the hairs of the stamens cultured on media R1, R3, R5 and M1 were similar to each other, and the distribution pattern
Table 5. Frequency of coloration occurred in the stamen hairs after 10 day culturing onfive different media (see Table l for the hormone concentrations) Medium no. R1 R3 R5 M1 0
Number of colored stamen hairs per flower* Class I Class II Class I I I O.5 3.6 7.6 2.6 0
9.6 13.7 14.2 21.8 0
23.5 9.9 24.0 80.7
*'Flower' means a cluster of five stamens plus ovary cultured. All the five stamens did not always survive.
260
C. N A G A S H I M A - I S H I I and S. I C H I K A W A
is d r a w n in Fig. 1 together with that observed in E x p e r i m e n t 1. T h e results are similar in b o t h experiments.
DISCUSSION
Culture of Tradescantia stamen hairs was first tried by PARCHMANt l ° ' l l ) using T. paludosa, to study h o r m o n a l effects on s t a m e n hair cells. H e observed m a x i m u m growth on m e d i u m containing 10-VM I A A a n d 1 0 - 6 M kinetin. T h e conditions allowed, however, division of s t a m e n hair cells to occur only onceJ TM I n the present study, an increase of as m a n y as 10 cells p e r hair was observed (Table 2). Cell division in Tradescantia s t a m e n hairs is k n o w n to occur mostly at the t e r m i n a l two cells of each hair. ~4~T h e 10 cell increase per hair means that 10 cell divisions on the average occurred p e r hair d u r i n g the culture on the most effective m e d i u m so far examined. T h e difference between PARCHMAN'S a n d our results seems to be due to different culture methods. T h e culture of b a r e stamens resulted in no a p p a r e n t g r o w t h in the p r e l i m i n a r y e x p e r i m e n t c o n d u c t e d before this study. C u l t u r i n g together with other floral organs (e.g. ovary and receptacle) a p p e a r s necessary for successful stamen culture. T h e r e has been no previous report on the coloration of cultured stamen hairs. Results from the present study suggest that coloration m a y be d e t e r m i n e d by h o r m o n a l combinations. Coloration of stamen hairs a n d frequency of colored hairs were most p r o m i n e n t on the m e d i u m which c o n t a i n e d 0.1 mg/1 I A A , 1.0mg/1 G A 3 a n d 0.1mg/1 kinetin (see Tables 1, 3 a n d 5). Sucrose concentrations higher than 4 % also seemed necessary, since the highest frequency of coloration occurred on m e d i u m M1 (see T a b l e s 1 a n d 5). T h e coloration did not occur at r a n d o m . I t was observed that all hairs of a stamen were colored while all hairs of other stamens of the same b u d r e m a i n e d colorless. T h u s s t a m e n - h a i r coloration seemed to be an all-or-nothing event. I n the n o r m a l in vivo flowers of this clone, s t a m e n - h a i r coloration is initiated 5 or 6 days before flowering t6) after c o m p l e t i n g r e p e a t e d divisions of the terminal cell of each hair. T h e completion of the
division is thus t h o u g h t to be a likely 'trigger' of coloration. Distribution patterns of colored cells (from the terminal-cell position to the tenth-cell position from the hair tip) as shown in Fig. 1 indicate that coloration starts at the s u b t e r m i n a l or terminal cells of hairs a n d proceeds to the basal portion. T h e postulate that c o m p l e t i o n of cell division is a 'trigger' of coloration does not c o n t r a d i c t these results, because the terminal a n d s u b t e r m i n a l cells directly receive information of division completion due to the n a t u r e in which cells increase in n u m b e r in stamen hairsJ 4) T h e results of the present study suggest the possibility of developing a successful in vitro culture technique of Tradescantia stamens. Such a technique might be useful a n d inexpensive for routine testing a n d screening of potential chemical and e n v i r o n m e n t a l mutagens.
Acknowledgement--We wish to express our sincere thanks to Prof. K. TSUNEWAKIof Kyoto University for his kindly guidance and encouragement extended to one of us (CNI) during the course of the present study.
REFERENCES 1. ICHIKAWA S. (1971) Somatic m u t a t i o n r a t e at low
levels of chronic gamma-ray exposures in Tradescantia stamen hairs. Jlap. J. Genet. 46, 371381. 2. IGItlKAWA S. (1972) Somatic mutation rate in Tradescantia stamen hairs at low radiation levels: Finding of low doubling doses of mutations, flap. J. Genet. 47, 411-421. 3. IeHIRAWA S. (1976) Tradescantia stamen hairs: A botanical mutagenicity testing system with a high sensitivity. Pages 52-56 in Laboratory manual for
East-Asian workshop on mutagenicity testing of chemicals, National Institute of Genetics, Misima. 4. ICHIKAWA S. and SPARROW A. H. (1967) Radiation-induced loss of reproductive integrity in the stamen hairs of polyploid series of Tradescantia species. Radiat. Bot. 7, 429~41. 5. ICHIKAWAS. and SPARROWA. H. (1968) The use of induced somatic mutations to study cell division rates in irradiated stamen hairs of Tradescantia virginiana L. Jlap. o7 Genet. 43, 57 63. 6. IcHn~AWAS., SPARROWA. H. and THOMPSONK. H. (1969) Morphologically abnormal cells, somatic mutations and loss of reproductive integrity in
TRADESCANTIA
7.
8.
9.
10.
11.
12.
STAMEN HAIRS CULTURED I N VITRO
irradiated Tradescantia stamen hairs. Radiat. Bot. 9, 195-211. IGHIKAWAS. and TAKAHASHIC. S. (1978) Somatic mutations in Tradescantia stamen hairs exposed to ethyl methanesulfonate. Envir. Expl. Bot. 18, 19-25. MERICLE L. W. and MERICLE R. P. (1967) Genetic nature of somatic mutations for flower color in Tradescantia, clone 02. Radiat. Bot. 7, 449464. NAYAR G. G. and SPARROW A. H. (1967) Radiation-induced somatic mutations and loss of reproductive integrity in Tradescantia stamen hairs. Radiat. Bot. 7, 257-267. PARCHMAN L. G. (1962) The effect of certain growth substances upon Tradescantia paludosa stamen hair cells cultured in vitro. Master's Thesis, Emory University. PARCHMANL. G. (1964) The morphogenesis of the stamen hairs of Tradescantia paludosa. Doctor's Thesis, Emory University. SPARROWA. H. and SCHAIRERL. A. (1974) The effects of chemical mutagens (EMS, DBE) and
13.
14.
15.
16.
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specific air pollutants (O3, SOz, NO2, N/O) on somatic mutation rate in Tradescantia. Text of talk at Symp. on The potential genetic effects of environmental pollutants on man, Moscow, U.S.S.R. Feb. 1974. SPARROWA. H., SCHAIRERL. A. and VILLALOBOSPIETRINI R. (1974) Comparison of somatic mutation rates induced in Tradescantia by chemical and physical mutagens. Murat. Res. 26, 265 276. SPARROW A. H., UNDERBRINK A. G. and RossI H. H. (1972) Mutations induced in Tradescantia by small doses of X-rays and neutrons: Analysis of dose-response curve. Science N. T. 176, 916-918. UNDERBRINKA. G., SCHAIRERL. A. and SPARROW A. H. (1973) Tradescantia stamen hairs: A radiobiological test system applicable to chemical nmtagenesis. Pages 171-207 in A. HOLLAENDER, ed. Chemical mutagens: Principles and methods for their detection, vol. 3, Plenum Press, New York. WHITER. P. (1963) The cultivation of animal andplant cells. Ronald Press, New York.