- Email: [email protected]
Tradescantia cytogenetic tests (root-tip mitosis, pollen mitosis, pollen mother-cell meiosis)
Mutation Research, 99 (1982) 293-302
293
Elsevier Biomedical Press
Tradescantia cytogenetic tests (root-tip mitosis, pollen
mitosis, pollen mother-cell meiosis) A report of the U.S. Environmental Protection Agency Gene-Tox Program * Te-Hsiu Ma Department of Bwlogwal Sctences and Institute for Environmental Management, Western Ilhnois University, Macomb, I L 61455 (U.S.A.)
(Received 7 April 1982) (Accepted 8 April 1982)
Summary 3 kinds of cytogenetic tests for screening of e n v i r o n m e n t a l m u t a g e n s were e s t a b l i s h e d for T r a d e s c a n t i a , namely, r o o t - t i p mitosis, pollen mitosis, a n d p o l l e n mother-ceU meiosis [ c o m m o n l y referred to as the T r a d e s c a n t i a - m i c r o n u c l e u s ( T r a d M C N ) test]. A l l these tests are technically simple, inexpensive, a n d can yield reliable results in a relatively short time ( 3 6 - 7 2 h). T h e r o o t - t i p mitosis test is suitable only for liquid agents, while pollen mitosis is suitable for b o t h liquid a n d gaseous agents. Pollen t u b e m i t o t i c c h r o m o s o m e s are extremely sensitive to mutagens; therefore, they are g o o d m a t e r i a l s for detecting very low c o n c e n t r a t i o n s of mutagens. Both r o o t - t i p mitosis a n d p o l l e n mitosis tests use c h r o m o s o m e a n d / o r c h r o m a t i d a b e r r a tions as e n d p o i n t s for scoring. The T r a d - M C N test is suitable for b o t h liquid a n d gaseous agents. I n addition, it is especially suitable for in situ m o n i t o r i n g of water
By acceptance of this article the publisher or recipient acknowledges the U.S. Government's right to retain a nonexclusive, royalty-free license in and to any copyright covering the article. * Work Group Report prepared for the Gene-Tox Program (Office of Toxic Substances, Office of Pesticides and Toxic Substances, U.S. Environmental Protection Agency, Washington, DC). The author is a member of the Gene-Tox Work Group on Higher Plant Genetic and Cytogenetic Assays. Although the review described in this article has been funded wholly or in part by the United States Environmental Protection Agency through Interagency Agreement DOE 40-1123-80, EPA No. 80-DX0953, to the Oak Ridge National Laboratory, it has not been subjected to the Agency's reqmred peer and policy review and, therefore, does not necessarily reflect the views of the Agency and no official endorsement should be inferred. The protocols stated, suggested use of the assay in a screening program, and research recommended should not be taken to represent Agency policy on these matters. 0165-1110/82/0000-0000/$02.75
© Elsevier Biomedical Press
294 and air pollutants. Of the 12 chemicals tested, 5-fluorouracil and 1,2-dibromoethane indicate that they are very potent mutagens based on the effective dosages used to produce a positive response. Sulfur dioxide, ethyl methanesulfonate, sodium azide, Phosdrin, and Bladex rank next in potency.
Tradescantia paludosa Anderson and Woodson (commonly called spiderwort) is a native species in the southern United States although a number of its relatives are indigenous to the eastern, southeastern, and midwestern states. It has 6 pairs of relatively large metacentric chromosomes in its pollen mother cells and root tips (Fig. 1) and a haploid number of 6 in pollen-tube nuclei (Fig. 2) and microspores. The extensive greenhouse cultivation of this species in the United States is mainly for the purpose of chromosome analyses in conjunction with radiation and environmental mutagen studies. T. paludosa clone No. 03 is a stout multibranched herbaceous plant. It tillers profusely in loose humus soil; thus, a large population of mature plants can be propagated in 40-60 days. The young inflorescences are composed of 16-20 buds in the form of a cyme. The blue or purplish-blue flowers are composed of 3 sepals, 3 petals, 6 anthers, and a pistil with a 3-celled ovary. Under natural conditions, it blooms in summer and early fall; flowers can be induced in short-day seasons, by supplementing with 6 - 8 h of artificial light. The greenhouse cultivated plants may attain the height of 40-60 cm and up to 20-30
Fig. 1. Mitotic chromosomesof Tradescantla root-Up meristem (2 n = 12).
295 ~ , } ~ '~:;,~ ~ "
"~ . . . . .
,~
~ "~ , ' ~-~ ~ ,,4
-
• ,,').
Fig. 2. M i t o t i c c h r o m o s o m e s o f T r a d e s c a n t l a p o l l e n t u b e ( n = 6).
plants per pot. Leaves are dark green, narrow, and long. The other commonly used Tradescantia, clone No. 02, has a similar morphology as clone No. 03 but has relatively slender stems and leaves. The Tradescantia clone No. 4430 is a hybrid of T. subacaulis and T. hirsutiflora. It has much longer hairs covering the surface of the leaves and stems and fewer branches than clone No. 03. Mitotic chromosomes of microspores of Tradescantia paludosa, and occasionally other species, have been utilized extensively for radiation studies, especially for •stablishing the mechanisms and types of chromosome and chromatid aberrations nduced by radiation. Unfortunately, microspore chromosomes have not been used extensively in studies of chemical mutagenesis because of difficulties in introducing chemical agents into the well-developed microspores. This difficulty is due mainly to the presence of the mucilaginous fluid in the anther that prevents foreign agents from entering the microspores. The mitotic chromosomes in the meristem of Tradescantia root tips have all the favorable features found in root meristems of Vicia and Allium. 12 relatively large metacentric chromosomes can be obtained easily from newly developed root tips. A large number of young roots can be produced from plant cuttings in about a week. These root tips have been used for chemical mutagen studies (Ahmed and Grant, 1972) in the same manner as described in Vica and Allium root-tip mitotic chromosome tests. The technique for in vitro culture of pollen tubes of Tradescantia was established by Bishop (1949) and improved by Conger (1953) and Ma (1967). The cultured pollen tube chromosomes have been used in radiation studies since the early 1950s. At about the same time, this system was used for chemical mutagen tests (Smith and Lotfy, 1954). A series of studies on SO 2 (Ma et al., 1973), hydroxyurea, and colchicine (Khan and Ma, 1974) were carried out. The advantages of this system are that the chromosomes are in haploid number (n = 6) and that the generative nucleus of the mature pollen is usually synchronized in the G 2 stage of interphase. Aberrations induced in the mature pollen are usually of the chromatid types. Chromosomes of mitotic pollen tube generative nuclei are extremely sensitive to gaseous impurities in the air. A near absolute clean air room or a glove box is necessary to grow the culture and conduct experiments successfully.
296 !
I
20/zm
1
ICN q
F~g. 3. Tetrad stage of meiotic pollen mother cells of Tradescantla. N. normal tetrad. MCN, mlcronuclei containing tetrads.
Meiotic chromosomes of pollen mother cells of Tradescantia stain intensely with the aceto-carmine squash technique if they are prepared from fresh pollen mother cells without fixation. Fixed pollen mother cells usually stain poorly and give fuzzy chromosome images. They are not practical for mutagen tests, which usually require a large number of samples fixed at the same time. Based on the fact that chromosome fragments in a meiotic process usually result in micronuclei in the tetrad stage (Fig. 3), a mutagen test system was developed using micronuclei as indicators of genetic damage (Ma et al., 1978). Because of the high sensitivity of early prophase I and the high degree of synchrony of early prophase I and tetrad stages, this test is highly effective. Preparation of tetrad slides and scoring of micronuclei are relatively easy and simple. Furthermore, this test has been proven to be suitable for both gaseous and liquid mutagens (Ma, 1979). 7 publications were selected as references for this report from the literature published in the past 40 years. Selection was based on adequate experimental design and valid data for negative or positive response to the agents tested. Only those tests dealing with a simple well-defined chemical compound were included. Tests of the effects of natural extracts or the secondary effect over the effect of a primary chemical agent were not included.
297
Test descriptions 3 different cytogenetic tests can be performed using Tradescantia, namely, root-tip mitosis, pollen mitosis, and pollen mother-cell meiosis. Only the general procedures of these tests are described below. For detailed accounts and some common well-known techniques in each of these tests, one should consult the references cited in this report.
Root-tip mitosis Tradescantia paludosa Sax clone No. 02 was frequently used for this test in the past, although other clones or species could serve the purpose. A large number of young roots can be obtained by maintaining the plant in a vermiculite bed with bottom heating (Ahmed and Grant, 1972) or by maintaining the cuttings in nutrient solution under artificial or natural light at 25-30°C and 70-80% relative humidity with constant aeration. Young roots (2-5 cm long) should be selected for experimentation. Treatment of the fast-dividing cells of the meristem can be carried out by immersing the root portion of the cuttings in an aqueous solution containing a known quantity of chemical for a given duration (6-24 h). Although these roots can tolerate a wide range of pH conditions (5-8), buffering of the solution is suggested whenever possible or appropriate. Generally an 8-40-h recovery period after the end of treatment is necessary for the chromosomes treated during their interphase (G 1, S, G 2) to arrive at metaphase of the same mitotic cycle or the second mitotic cycle, respectively. For more reliable quantitative results and easy scoring, 8-h post-treatment fixation samples should be used to obtain chromatid aberrations. Types of chromatid aberrations and the scoring sheet are shown in Appendix 1 of the report on Vicia cytogenetic tests (Ma, 1982). In order to accumulate a large number of metaphase figures, colchicine should be applied to the roots before fixation. This is accomplished efficiently by using bubbling air to tumble the excised root tips (about 1-2 cm long) in 0.2% colchicine solution in a small vial for 3 - 4 h. Carnoy's fluid (aceto-alcohol 1:3 ratio) is one of the common fixatives used. Other fixatives are Newcomer's, Gate's, Navashin's, and Ford's. A 24-h fixation period is usually sufficient, and the fixed roots can be stored in 70% ethanol in a refrigerator for several weeks. The aceto-carmine squash method is a simple and efficient technique for slide preparation. For greater clarity of the chromosomes and the aberrant chromosome configurations, the Feulgen reaction is recommended. Pollen mitosis Pollen grains of Tradescantia paludosa clone No. 03 have been used frequently for in vitro culture of pollen tubes. In order to create a monolayer of pollen to facilitate the uniform exposure to the gaseous agents and to distribute the pollen evenly on the culture medium, pollen collected from fully opened flowers should be desiccated for at least 4 h in the dark. The dry pollen should be sown on lactose (12.0%) agar (1.5%) medium by means of a camel hair brush (Ma, 1967). If a gaseous agent is applied to the dry pollen or to the pollen at the very beginning of germination, the chromosomes of G 2 stage would suffer damage, and chromatid aberrations would be
298 anticipated (Ma et al., 1973). The liquid agents to be tested should be dissolved in water and incorporated in the medium (Khan and Ma, 1974) with colchicine (0.02%) at the time that the medium is prepared. The culture must be incubated in a refrigerated incubator at 22 ± I°C in the dark. The pollen tubes, together with the chromosomes of the generative nuclei, are fixed in Carnoy's fluid (or another fixative) after 16-20 h growth. Chromosomes can be stained with aceto-carmine or by means of the Feulgen reaction after removal of the excess medium from the slide (Ma, 1967). Frequencies of various types of chromatid aberrations can be scored under 1000 X magnification and expressed in terms of breaks per 100 cells. Pollen mother-cell melosts
Young inflorescences of Tradescantia can be produced year round in growth chambers (Underbrink et al., 1973) or in greenhouses with supplemented day length during short-day seasons to facilitate continuous experimentation. Since the chromosomes of the pollen mother cells, when fixed, cannot be clearly stained to show the typical chromosome or chromatid aberrations, it is not efficient or reliable to use these chromosome aberrations as indicators of genetic damage. Quantitative data are obtained, however, by using micronuclei in the tetrad stage as the indicator of genetic damage. This newly established test system (Ma et al., 1978; Ma, 1979) can be used for environmental mutagen testing as well as for in situ monitoring. By taking advantage of the high sensitivity of the early prophase I chromosomes and their high degree of synchrony, treatment can be applied to a large population of sensitive cells at the same meiotic stage. Thus, chromosome breaks can be observed as micronuclei in the synchronized tetrads. Practically, this is accomplished by exposing the excised young inflorescences to gaseous agents in a confined chamber or allowing cuttings to absorb liquid agents from a prepared solution (Ma, 1979). Gaseous agents enter the buds and then the anthers; liquid agents can be introduced into the pollen mother cells through the stem, peduncle, and pedicel of the cuttings. After either of these 2 types of treatment, a recovery (meiotic) period of 2 4 - 3 0 h would allow the damaged chromosomes of early prophase I to arrive at the tetrad stage, when micronuclei are easily scored. Micronuclei are usually formed from acentric fragments or clumped chromosomes. Microslides of the tetrad stage are prepared using the aceto-carmine squash method. Scoring of micronuclei among 1000-1500 tetrads provides adequate data to show the magnitude of chromosome damage.
Interpretation of data In the 7 publications reviewed, only 12 chemicals were tested in the Tradescantia cytogenetic assays. Root-tip mitotic chromosome aberration tests were used to detect the effects of Phosdrin and Bladex (pesticides) at gradient concentrations or in a series of increasing treatment duration. Ethyl methanesulfonate (EMS) was used as a positive control in this series of experiments (Ahmed and Grant, 1972). Mutagen
299 tests on methyl chloride, ethylene oxide, and ketene, using pollen-tube mitotic chromatid aberration, were conducted under 2-3 different dosages, while SO2 and hydroxyurea were tested at a single dose for the purpose of determining seasonal variation. Tests using meiotic chromosomes of pollen mother cells were applied to known mutagens such as 5-fluorouracil (Bempong and Trower, 1976), 1,2-dibromoethane (Ma et al., 1978), EMS, sodium azide, and hydrazoic acid (Ma, 1979). Of these chemicals, 1,2-dibromoethane showed clear linear dose response in the Tradescantia-micronucleus test and indicated a direct dose response in most cases. Meiotic chromosome aberrations and anomalous chromosome associations were used as the end points in the test of 5-fluorouracil (Bempong and Trower, 1976). All 3 kinds of these cytogenetic tests are suitable for screening of potential mutagens. The quantitative data should be presented in terms of number of aberrations per 100 cells in mitotic root-tip or pollen-tube tests. In the case of micronuclei, the frequencies should be expressed in terms of number of micronuclei per 100 tetrads. A minimum number of 300 cells or 1500 tetrads should be scored from each of the treated and control groups for the frequencies of chromosome or chromatid aberrations in mitotic cells and micronuclei in tetrads, respectively. X-Rays (physical), EMS (liquid chemical), and hydrazoic acid (gaseous chemical) are suitable for positive control agents, and water is suitable as a negative control agent. Statistical analysis should be applied to show the level of significance between treated and control groups. In order to obtain more reliable cause-effect relationships, a series of increasing doses should be applied to establish a dose-response curve. Doses of gaseous agents should be expressed in percentages or parts per million (ppm) units, and those in liquid form should be expressed in molarity, percentage, or ppm units. All measurements of length (cm), volume (ml), and weight (g) should be expressed in metric units; temperature should be given in degrees Celsius (°C) and pressure in millimeters of mercury. All chemicals tested with the Tradescantia cytogenetic tests that show a positive response could be considered as potential mutagens. Further tests in mammalian systems should be performed for possible carcinogenic and teratogenic effects.
Test pedormance A total of 12 chemicals including 8 chemical classes were tested in the Tradescantia cytogenetic system (Table 1). Because of the small number of chemicals tested under 3 different types of tests, no conclusion can be drawn on chemicals or chemical classes which give anomalous results. By comparison, 1,2-dibromoethane, hydrazoic acid, and hydroxyurea are highly potent mutagens. Their mutagenic activities seem to be greater than those of EMS. In general, it is probably true that all Tradescantia cytogenetic tests are suitable for water-soluble agents. The pollen mitotic chromatid aberration test is particularly efficient in testing extremely low concentrations of gaseous agents. The Trad-MCN test using pollen mother cells is suitable for gaseous as well as liquid agents with relatively high efficiency. The simplicity in slide preparation and scoring procedure in the Trad-MCN test makes it
300
0
d
Q
0 E ,o
- -
~.
~
-
~ E E
o
r~
r~
r~ r~ r~
r~
+
-t-
-t- -I- -t-
-I-
E
0
r~
-I-
-l--k-t-
-I-
-I-
-I--F
o 2
~z
~Z
~.
.~
o "~ ~
~
~
•~
E '~ .o ~, ~ ~
E ~
o "~
-
z
ZZ
~ ..~ ....
.~.
~
.~ ..~ ~
.~
_
~=
z
<
,_
z
~
O~ 0 " ~ ,~ "i"
~ 8..~ J
~
301
suitable for large-scale screening of potential mutagens and for in situ monitoring of pollutants. No comparison can be made between Tradescantia cytogenetic tests and mammalian tests in reference to carcinogenicity because chromosome damage in plants cannot be directly equated with carcinogenesis, and there are insufficient data, at this time, to deduce the reliable relative efficiencies between these two different end points.
Conclusion All of these Tradescantia cytogenetic tests can be used as preliminary screening bioassays for mutagens. The major strengths and weaknesses of each of these 3 bioassays are elaborated below.
Root-tip mitosis Root-tip mitotic chromosome or chromatid aberrations can be scored easily and yield reliable qualitative results. The ratio between single chromatid break and chromatid exchange frequencies has been used to differentiate the mechanism of breakage induced by different agents. Chromosomes are relatively large in size and small in number. They are also potential experimental material for sister-chromatid exchange studies. Above all, the roots can be obtained in large quantities all year round with minimum expense. The asynchronous meristematic cells result in relatively low efficiency of this test as compared with the test that uses synchronous cells. Pollen mitosis
This test uses pollen grains as free cells in culture medium. Thus, the experimental condition can be controlled to the fullest extent. Small numbers of chromosomes ( n - - 6 ) simplify the task of chromatid aberration analysis. The pollen tube has a very short mitotic cycle (16-20h) in its generative nucleus, thus reducing the experimental time to a minimum. The weakness in this system is that the pollen-tube culture is extremely sensitive to the toxicity of environmental agents in gaseous or liquid form. Chromosomes are relatively small, and 1000 × magnification is needed to score the chromatid aberrations. Pollen mother-cell meiosis
This is a more sensitive system in detecting mutagens than is the system of mitotic chromosomes of root tips. Unfortunately, the occurrence of chromosome aberrations in metaphase I is not a reliable indicator of genetic damage because of the unclear chromosome image. The Trad-MCN test can yield reliable quantitative data in terms of number of micronuclei per 100 tetrads. Preparation of slides and scoring of micronuclei frequencies are relatively easy. This test can be used to determine the clastogenicity of gaseous and liquid agents, and it can also be used for in situ
302 m o n i t o r i n g o f p o l l u t i o n sites. E a c h test c a n be a c c o m p l i s h e d in 2 4 - 4 8 h w i t h r e l a t i v e l y l o w cost. T e s t results c a n b e u s e d to c r o s s c h e c k the results o f T r a d e s c a n t i a s t a m i n a l h a i r m u t a t i o n studies. T h u s , the c o r r e l a t i o n b e t w e e n : o m a t i c g e n e m u t a t i o n a n d c h r o m o s o m e d a m a g e c a n b e e s t a b l i s h e d . O n e c h a r a c t e r i s t i c to be c o n s i d e r e d as a w e a k n e s s o f this test is the h i g h s e n s i t i v i t y o f the m e i o t i c c h r o m o s o m e s to f o r e i g n agents. A n o v e r d o s e o f t e n i n d u c e s m e i o t i c d e l a y o r s e v e r e p h y s i o l o g i c a l d a m a g e , w h i c h m a k e s testing v e r y difficult.
References Ahmed, M., and W.F. Grant (1972) Cytological effect of the pesticides Phosdrin and Bladex on Tradescantia and Vtclafaba, Can. J. Genet. Cytol., 14, 157-165. Bempong. M.A.. and E.C. Trower (1976) Cytogenetic effects of 5-fluorouracil, V. Induced meiotic abnormalities in Tradescantta paludosa, Bull. Torrey Bot. Club, 103, 109-116. Bishop, D.J. (1949) Pollen tube culture on lactose medium. Stain Technol., 24, 9-12. Conger, A.D. (1953) Culture of pollen tubes for chromosomal analysls at the pollen tube division, Stare Technol., 28, 289-293. Khan, S.H., and T.H. Ma (1974) Hydroxyurea enhanced chromatld aberrations in Tradescantia pollen tubes and seasonal vanation of aberration rates, Mutation Res., 15, 33-38. Ma. T.-H. (1967) Thin-layer lactose-agar for pollen tube culture of Tradescantla to enhance planar distribution of chromosomes, Stain Technol., 42, 285-291. Ma, T.-H. (1979) Micronuclei induced by X-rays and chemical mutagens m meiotic pollen mother cells of Tradescantia, A pronusing mutagen test system, Mutation Res., 64. 307-313. Ma, T.-H. (1982) Vicia cytogenetic test for environmental mutagens, A report of the U.S. Environmental Protection Agency Gene-Tox Program, Mutation Res., 99, 257-271. Ma, T.-H., D. Isbandi, S.H. Khan and Y.S. Tseng (1973) Low level SO2 enhanced chromatid aberrations in Tradescantia pollen tubes and seasonal variation of the aberration rates, Mutation Res., 21, 93-100. Ma, T-H., A.H. Sparrow, L.A. Schairer and A.F. Nauman (1978) Effect of 1,2-dibromoethane (DBE) on meiotic chromosomes of Tradescantia, Mutation Res., 58, 251-258. Smith, H.H., and T.A. Lotfy (1954) Comparative effect of certain chemicals on Tradescantla chromosomes as observed at pollen tube mitosis, Am. J. Bot., 41,589-593. Underbrink, A.G., L.A. Schairer and A.H. Sparrow (1973) Tradescantia stamen hairs: A radiological test system applicable to chemical mutagenesis, in: A. Hollaender (Ed.), Chemical Mutagens, Principles and Methods for their Detection, Vol. 3, Plenum, New York, pp. 171-207.
293
Elsevier Biomedical Press
Tradescantia cytogenetic tests (root-tip mitosis, pollen
mitosis, pollen mother-cell meiosis) A report of the U.S. Environmental Protection Agency Gene-Tox Program * Te-Hsiu Ma Department of Bwlogwal Sctences and Institute for Environmental Management, Western Ilhnois University, Macomb, I L 61455 (U.S.A.)
(Received 7 April 1982) (Accepted 8 April 1982)
Summary 3 kinds of cytogenetic tests for screening of e n v i r o n m e n t a l m u t a g e n s were e s t a b l i s h e d for T r a d e s c a n t i a , namely, r o o t - t i p mitosis, pollen mitosis, a n d p o l l e n mother-ceU meiosis [ c o m m o n l y referred to as the T r a d e s c a n t i a - m i c r o n u c l e u s ( T r a d M C N ) test]. A l l these tests are technically simple, inexpensive, a n d can yield reliable results in a relatively short time ( 3 6 - 7 2 h). T h e r o o t - t i p mitosis test is suitable only for liquid agents, while pollen mitosis is suitable for b o t h liquid a n d gaseous agents. Pollen t u b e m i t o t i c c h r o m o s o m e s are extremely sensitive to mutagens; therefore, they are g o o d m a t e r i a l s for detecting very low c o n c e n t r a t i o n s of mutagens. Both r o o t - t i p mitosis a n d p o l l e n mitosis tests use c h r o m o s o m e a n d / o r c h r o m a t i d a b e r r a tions as e n d p o i n t s for scoring. The T r a d - M C N test is suitable for b o t h liquid a n d gaseous agents. I n addition, it is especially suitable for in situ m o n i t o r i n g of water
By acceptance of this article the publisher or recipient acknowledges the U.S. Government's right to retain a nonexclusive, royalty-free license in and to any copyright covering the article. * Work Group Report prepared for the Gene-Tox Program (Office of Toxic Substances, Office of Pesticides and Toxic Substances, U.S. Environmental Protection Agency, Washington, DC). The author is a member of the Gene-Tox Work Group on Higher Plant Genetic and Cytogenetic Assays. Although the review described in this article has been funded wholly or in part by the United States Environmental Protection Agency through Interagency Agreement DOE 40-1123-80, EPA No. 80-DX0953, to the Oak Ridge National Laboratory, it has not been subjected to the Agency's reqmred peer and policy review and, therefore, does not necessarily reflect the views of the Agency and no official endorsement should be inferred. The protocols stated, suggested use of the assay in a screening program, and research recommended should not be taken to represent Agency policy on these matters. 0165-1110/82/0000-0000/$02.75
© Elsevier Biomedical Press
294 and air pollutants. Of the 12 chemicals tested, 5-fluorouracil and 1,2-dibromoethane indicate that they are very potent mutagens based on the effective dosages used to produce a positive response. Sulfur dioxide, ethyl methanesulfonate, sodium azide, Phosdrin, and Bladex rank next in potency.
Tradescantia paludosa Anderson and Woodson (commonly called spiderwort) is a native species in the southern United States although a number of its relatives are indigenous to the eastern, southeastern, and midwestern states. It has 6 pairs of relatively large metacentric chromosomes in its pollen mother cells and root tips (Fig. 1) and a haploid number of 6 in pollen-tube nuclei (Fig. 2) and microspores. The extensive greenhouse cultivation of this species in the United States is mainly for the purpose of chromosome analyses in conjunction with radiation and environmental mutagen studies. T. paludosa clone No. 03 is a stout multibranched herbaceous plant. It tillers profusely in loose humus soil; thus, a large population of mature plants can be propagated in 40-60 days. The young inflorescences are composed of 16-20 buds in the form of a cyme. The blue or purplish-blue flowers are composed of 3 sepals, 3 petals, 6 anthers, and a pistil with a 3-celled ovary. Under natural conditions, it blooms in summer and early fall; flowers can be induced in short-day seasons, by supplementing with 6 - 8 h of artificial light. The greenhouse cultivated plants may attain the height of 40-60 cm and up to 20-30
Fig. 1. Mitotic chromosomesof Tradescantla root-Up meristem (2 n = 12).
295 ~ , } ~ '~:;,~ ~ "
"~ . . . . .
,~
~ "~ , ' ~-~ ~ ,,4
-
• ,,').
Fig. 2. M i t o t i c c h r o m o s o m e s o f T r a d e s c a n t l a p o l l e n t u b e ( n = 6).
plants per pot. Leaves are dark green, narrow, and long. The other commonly used Tradescantia, clone No. 02, has a similar morphology as clone No. 03 but has relatively slender stems and leaves. The Tradescantia clone No. 4430 is a hybrid of T. subacaulis and T. hirsutiflora. It has much longer hairs covering the surface of the leaves and stems and fewer branches than clone No. 03. Mitotic chromosomes of microspores of Tradescantia paludosa, and occasionally other species, have been utilized extensively for radiation studies, especially for •stablishing the mechanisms and types of chromosome and chromatid aberrations nduced by radiation. Unfortunately, microspore chromosomes have not been used extensively in studies of chemical mutagenesis because of difficulties in introducing chemical agents into the well-developed microspores. This difficulty is due mainly to the presence of the mucilaginous fluid in the anther that prevents foreign agents from entering the microspores. The mitotic chromosomes in the meristem of Tradescantia root tips have all the favorable features found in root meristems of Vicia and Allium. 12 relatively large metacentric chromosomes can be obtained easily from newly developed root tips. A large number of young roots can be produced from plant cuttings in about a week. These root tips have been used for chemical mutagen studies (Ahmed and Grant, 1972) in the same manner as described in Vica and Allium root-tip mitotic chromosome tests. The technique for in vitro culture of pollen tubes of Tradescantia was established by Bishop (1949) and improved by Conger (1953) and Ma (1967). The cultured pollen tube chromosomes have been used in radiation studies since the early 1950s. At about the same time, this system was used for chemical mutagen tests (Smith and Lotfy, 1954). A series of studies on SO 2 (Ma et al., 1973), hydroxyurea, and colchicine (Khan and Ma, 1974) were carried out. The advantages of this system are that the chromosomes are in haploid number (n = 6) and that the generative nucleus of the mature pollen is usually synchronized in the G 2 stage of interphase. Aberrations induced in the mature pollen are usually of the chromatid types. Chromosomes of mitotic pollen tube generative nuclei are extremely sensitive to gaseous impurities in the air. A near absolute clean air room or a glove box is necessary to grow the culture and conduct experiments successfully.
296 !
I
20/zm
1
ICN q
F~g. 3. Tetrad stage of meiotic pollen mother cells of Tradescantla. N. normal tetrad. MCN, mlcronuclei containing tetrads.
Meiotic chromosomes of pollen mother cells of Tradescantia stain intensely with the aceto-carmine squash technique if they are prepared from fresh pollen mother cells without fixation. Fixed pollen mother cells usually stain poorly and give fuzzy chromosome images. They are not practical for mutagen tests, which usually require a large number of samples fixed at the same time. Based on the fact that chromosome fragments in a meiotic process usually result in micronuclei in the tetrad stage (Fig. 3), a mutagen test system was developed using micronuclei as indicators of genetic damage (Ma et al., 1978). Because of the high sensitivity of early prophase I and the high degree of synchrony of early prophase I and tetrad stages, this test is highly effective. Preparation of tetrad slides and scoring of micronuclei are relatively easy and simple. Furthermore, this test has been proven to be suitable for both gaseous and liquid mutagens (Ma, 1979). 7 publications were selected as references for this report from the literature published in the past 40 years. Selection was based on adequate experimental design and valid data for negative or positive response to the agents tested. Only those tests dealing with a simple well-defined chemical compound were included. Tests of the effects of natural extracts or the secondary effect over the effect of a primary chemical agent were not included.
297
Test descriptions 3 different cytogenetic tests can be performed using Tradescantia, namely, root-tip mitosis, pollen mitosis, and pollen mother-cell meiosis. Only the general procedures of these tests are described below. For detailed accounts and some common well-known techniques in each of these tests, one should consult the references cited in this report.
Root-tip mitosis Tradescantia paludosa Sax clone No. 02 was frequently used for this test in the past, although other clones or species could serve the purpose. A large number of young roots can be obtained by maintaining the plant in a vermiculite bed with bottom heating (Ahmed and Grant, 1972) or by maintaining the cuttings in nutrient solution under artificial or natural light at 25-30°C and 70-80% relative humidity with constant aeration. Young roots (2-5 cm long) should be selected for experimentation. Treatment of the fast-dividing cells of the meristem can be carried out by immersing the root portion of the cuttings in an aqueous solution containing a known quantity of chemical for a given duration (6-24 h). Although these roots can tolerate a wide range of pH conditions (5-8), buffering of the solution is suggested whenever possible or appropriate. Generally an 8-40-h recovery period after the end of treatment is necessary for the chromosomes treated during their interphase (G 1, S, G 2) to arrive at metaphase of the same mitotic cycle or the second mitotic cycle, respectively. For more reliable quantitative results and easy scoring, 8-h post-treatment fixation samples should be used to obtain chromatid aberrations. Types of chromatid aberrations and the scoring sheet are shown in Appendix 1 of the report on Vicia cytogenetic tests (Ma, 1982). In order to accumulate a large number of metaphase figures, colchicine should be applied to the roots before fixation. This is accomplished efficiently by using bubbling air to tumble the excised root tips (about 1-2 cm long) in 0.2% colchicine solution in a small vial for 3 - 4 h. Carnoy's fluid (aceto-alcohol 1:3 ratio) is one of the common fixatives used. Other fixatives are Newcomer's, Gate's, Navashin's, and Ford's. A 24-h fixation period is usually sufficient, and the fixed roots can be stored in 70% ethanol in a refrigerator for several weeks. The aceto-carmine squash method is a simple and efficient technique for slide preparation. For greater clarity of the chromosomes and the aberrant chromosome configurations, the Feulgen reaction is recommended. Pollen mitosis Pollen grains of Tradescantia paludosa clone No. 03 have been used frequently for in vitro culture of pollen tubes. In order to create a monolayer of pollen to facilitate the uniform exposure to the gaseous agents and to distribute the pollen evenly on the culture medium, pollen collected from fully opened flowers should be desiccated for at least 4 h in the dark. The dry pollen should be sown on lactose (12.0%) agar (1.5%) medium by means of a camel hair brush (Ma, 1967). If a gaseous agent is applied to the dry pollen or to the pollen at the very beginning of germination, the chromosomes of G 2 stage would suffer damage, and chromatid aberrations would be
298 anticipated (Ma et al., 1973). The liquid agents to be tested should be dissolved in water and incorporated in the medium (Khan and Ma, 1974) with colchicine (0.02%) at the time that the medium is prepared. The culture must be incubated in a refrigerated incubator at 22 ± I°C in the dark. The pollen tubes, together with the chromosomes of the generative nuclei, are fixed in Carnoy's fluid (or another fixative) after 16-20 h growth. Chromosomes can be stained with aceto-carmine or by means of the Feulgen reaction after removal of the excess medium from the slide (Ma, 1967). Frequencies of various types of chromatid aberrations can be scored under 1000 X magnification and expressed in terms of breaks per 100 cells. Pollen mother-cell melosts
Young inflorescences of Tradescantia can be produced year round in growth chambers (Underbrink et al., 1973) or in greenhouses with supplemented day length during short-day seasons to facilitate continuous experimentation. Since the chromosomes of the pollen mother cells, when fixed, cannot be clearly stained to show the typical chromosome or chromatid aberrations, it is not efficient or reliable to use these chromosome aberrations as indicators of genetic damage. Quantitative data are obtained, however, by using micronuclei in the tetrad stage as the indicator of genetic damage. This newly established test system (Ma et al., 1978; Ma, 1979) can be used for environmental mutagen testing as well as for in situ monitoring. By taking advantage of the high sensitivity of the early prophase I chromosomes and their high degree of synchrony, treatment can be applied to a large population of sensitive cells at the same meiotic stage. Thus, chromosome breaks can be observed as micronuclei in the synchronized tetrads. Practically, this is accomplished by exposing the excised young inflorescences to gaseous agents in a confined chamber or allowing cuttings to absorb liquid agents from a prepared solution (Ma, 1979). Gaseous agents enter the buds and then the anthers; liquid agents can be introduced into the pollen mother cells through the stem, peduncle, and pedicel of the cuttings. After either of these 2 types of treatment, a recovery (meiotic) period of 2 4 - 3 0 h would allow the damaged chromosomes of early prophase I to arrive at the tetrad stage, when micronuclei are easily scored. Micronuclei are usually formed from acentric fragments or clumped chromosomes. Microslides of the tetrad stage are prepared using the aceto-carmine squash method. Scoring of micronuclei among 1000-1500 tetrads provides adequate data to show the magnitude of chromosome damage.
Interpretation of data In the 7 publications reviewed, only 12 chemicals were tested in the Tradescantia cytogenetic assays. Root-tip mitotic chromosome aberration tests were used to detect the effects of Phosdrin and Bladex (pesticides) at gradient concentrations or in a series of increasing treatment duration. Ethyl methanesulfonate (EMS) was used as a positive control in this series of experiments (Ahmed and Grant, 1972). Mutagen
299 tests on methyl chloride, ethylene oxide, and ketene, using pollen-tube mitotic chromatid aberration, were conducted under 2-3 different dosages, while SO2 and hydroxyurea were tested at a single dose for the purpose of determining seasonal variation. Tests using meiotic chromosomes of pollen mother cells were applied to known mutagens such as 5-fluorouracil (Bempong and Trower, 1976), 1,2-dibromoethane (Ma et al., 1978), EMS, sodium azide, and hydrazoic acid (Ma, 1979). Of these chemicals, 1,2-dibromoethane showed clear linear dose response in the Tradescantia-micronucleus test and indicated a direct dose response in most cases. Meiotic chromosome aberrations and anomalous chromosome associations were used as the end points in the test of 5-fluorouracil (Bempong and Trower, 1976). All 3 kinds of these cytogenetic tests are suitable for screening of potential mutagens. The quantitative data should be presented in terms of number of aberrations per 100 cells in mitotic root-tip or pollen-tube tests. In the case of micronuclei, the frequencies should be expressed in terms of number of micronuclei per 100 tetrads. A minimum number of 300 cells or 1500 tetrads should be scored from each of the treated and control groups for the frequencies of chromosome or chromatid aberrations in mitotic cells and micronuclei in tetrads, respectively. X-Rays (physical), EMS (liquid chemical), and hydrazoic acid (gaseous chemical) are suitable for positive control agents, and water is suitable as a negative control agent. Statistical analysis should be applied to show the level of significance between treated and control groups. In order to obtain more reliable cause-effect relationships, a series of increasing doses should be applied to establish a dose-response curve. Doses of gaseous agents should be expressed in percentages or parts per million (ppm) units, and those in liquid form should be expressed in molarity, percentage, or ppm units. All measurements of length (cm), volume (ml), and weight (g) should be expressed in metric units; temperature should be given in degrees Celsius (°C) and pressure in millimeters of mercury. All chemicals tested with the Tradescantia cytogenetic tests that show a positive response could be considered as potential mutagens. Further tests in mammalian systems should be performed for possible carcinogenic and teratogenic effects.
Test pedormance A total of 12 chemicals including 8 chemical classes were tested in the Tradescantia cytogenetic system (Table 1). Because of the small number of chemicals tested under 3 different types of tests, no conclusion can be drawn on chemicals or chemical classes which give anomalous results. By comparison, 1,2-dibromoethane, hydrazoic acid, and hydroxyurea are highly potent mutagens. Their mutagenic activities seem to be greater than those of EMS. In general, it is probably true that all Tradescantia cytogenetic tests are suitable for water-soluble agents. The pollen mitotic chromatid aberration test is particularly efficient in testing extremely low concentrations of gaseous agents. The Trad-MCN test using pollen mother cells is suitable for gaseous as well as liquid agents with relatively high efficiency. The simplicity in slide preparation and scoring procedure in the Trad-MCN test makes it
300
0
d
Q
0 E ,o
- -
~.
~
-
~ E E
o
r~
r~
r~ r~ r~
r~
+
-t-
-t- -I- -t-
-I-
E
0
r~
-I-
-l--k-t-
-I-
-I-
-I--F
o 2
~z
~Z
~.
.~
o "~ ~
~
~
•~
E '~ .o ~, ~ ~
E ~
o "~
-
z
ZZ
~ ..~ ....
.~.
~
.~ ..~ ~
.~
_
~=
z
<
,_
z
~
O~ 0 " ~ ,~ "i"
~ 8..~ J
~
301
suitable for large-scale screening of potential mutagens and for in situ monitoring of pollutants. No comparison can be made between Tradescantia cytogenetic tests and mammalian tests in reference to carcinogenicity because chromosome damage in plants cannot be directly equated with carcinogenesis, and there are insufficient data, at this time, to deduce the reliable relative efficiencies between these two different end points.
Conclusion All of these Tradescantia cytogenetic tests can be used as preliminary screening bioassays for mutagens. The major strengths and weaknesses of each of these 3 bioassays are elaborated below.
Root-tip mitosis Root-tip mitotic chromosome or chromatid aberrations can be scored easily and yield reliable qualitative results. The ratio between single chromatid break and chromatid exchange frequencies has been used to differentiate the mechanism of breakage induced by different agents. Chromosomes are relatively large in size and small in number. They are also potential experimental material for sister-chromatid exchange studies. Above all, the roots can be obtained in large quantities all year round with minimum expense. The asynchronous meristematic cells result in relatively low efficiency of this test as compared with the test that uses synchronous cells. Pollen mitosis
This test uses pollen grains as free cells in culture medium. Thus, the experimental condition can be controlled to the fullest extent. Small numbers of chromosomes ( n - - 6 ) simplify the task of chromatid aberration analysis. The pollen tube has a very short mitotic cycle (16-20h) in its generative nucleus, thus reducing the experimental time to a minimum. The weakness in this system is that the pollen-tube culture is extremely sensitive to the toxicity of environmental agents in gaseous or liquid form. Chromosomes are relatively small, and 1000 × magnification is needed to score the chromatid aberrations. Pollen mother-cell meiosis
This is a more sensitive system in detecting mutagens than is the system of mitotic chromosomes of root tips. Unfortunately, the occurrence of chromosome aberrations in metaphase I is not a reliable indicator of genetic damage because of the unclear chromosome image. The Trad-MCN test can yield reliable quantitative data in terms of number of micronuclei per 100 tetrads. Preparation of slides and scoring of micronuclei frequencies are relatively easy. This test can be used to determine the clastogenicity of gaseous and liquid agents, and it can also be used for in situ
302 m o n i t o r i n g o f p o l l u t i o n sites. E a c h test c a n be a c c o m p l i s h e d in 2 4 - 4 8 h w i t h r e l a t i v e l y l o w cost. T e s t results c a n b e u s e d to c r o s s c h e c k the results o f T r a d e s c a n t i a s t a m i n a l h a i r m u t a t i o n studies. T h u s , the c o r r e l a t i o n b e t w e e n : o m a t i c g e n e m u t a t i o n a n d c h r o m o s o m e d a m a g e c a n b e e s t a b l i s h e d . O n e c h a r a c t e r i s t i c to be c o n s i d e r e d as a w e a k n e s s o f this test is the h i g h s e n s i t i v i t y o f the m e i o t i c c h r o m o s o m e s to f o r e i g n agents. A n o v e r d o s e o f t e n i n d u c e s m e i o t i c d e l a y o r s e v e r e p h y s i o l o g i c a l d a m a g e , w h i c h m a k e s testing v e r y difficult.
References Ahmed, M., and W.F. Grant (1972) Cytological effect of the pesticides Phosdrin and Bladex on Tradescantia and Vtclafaba, Can. J. Genet. Cytol., 14, 157-165. Bempong. M.A.. and E.C. Trower (1976) Cytogenetic effects of 5-fluorouracil, V. Induced meiotic abnormalities in Tradescantta paludosa, Bull. Torrey Bot. Club, 103, 109-116. Bishop, D.J. (1949) Pollen tube culture on lactose medium. Stain Technol., 24, 9-12. Conger, A.D. (1953) Culture of pollen tubes for chromosomal analysls at the pollen tube division, Stare Technol., 28, 289-293. Khan, S.H., and T.H. Ma (1974) Hydroxyurea enhanced chromatld aberrations in Tradescantia pollen tubes and seasonal vanation of aberration rates, Mutation Res., 15, 33-38. Ma. T.-H. (1967) Thin-layer lactose-agar for pollen tube culture of Tradescantla to enhance planar distribution of chromosomes, Stain Technol., 42, 285-291. Ma, T.-H. (1979) Micronuclei induced by X-rays and chemical mutagens m meiotic pollen mother cells of Tradescantia, A pronusing mutagen test system, Mutation Res., 64. 307-313. Ma, T.-H. (1982) Vicia cytogenetic test for environmental mutagens, A report of the U.S. Environmental Protection Agency Gene-Tox Program, Mutation Res., 99, 257-271. Ma, T.-H., D. Isbandi, S.H. Khan and Y.S. Tseng (1973) Low level SO2 enhanced chromatid aberrations in Tradescantia pollen tubes and seasonal variation of the aberration rates, Mutation Res., 21, 93-100. Ma, T-H., A.H. Sparrow, L.A. Schairer and A.F. Nauman (1978) Effect of 1,2-dibromoethane (DBE) on meiotic chromosomes of Tradescantia, Mutation Res., 58, 251-258. Smith, H.H., and T.A. Lotfy (1954) Comparative effect of certain chemicals on Tradescantla chromosomes as observed at pollen tube mitosis, Am. J. Bot., 41,589-593. Underbrink, A.G., L.A. Schairer and A.H. Sparrow (1973) Tradescantia stamen hairs: A radiological test system applicable to chemical mutagenesis, in: A. Hollaender (Ed.), Chemical Mutagens, Principles and Methods for their Detection, Vol. 3, Plenum, New York, pp. 171-207.