The selection of resistance mutants from cultured plant cells

91

Mutation Research, 72 (1980) 91--100

© Elsevier/North-HollandBiomedicalPress

THE SELECTION OF RESISTANCE MUTANTS FROM CULTURED PLANT CELLS

ROBERT B. HORSCH and GARY E. JONES Department of Botany and Plant Sciences, Cell Interaction Group, University of California, Riverside, CA 92521 (U.S.A.)

(Received 27 November 1979) (Revision received 6 March 1980) (Accepted 1 April 1980)

Summary We have determined the influence of several factors on the recovery of resistance mutants from populations of cultured plant cells. Cells of strains of Haplopappus gracilis resistant to the metabolite~analognes 8-azaguanine, 6~azauracil, or 5-hydroxylysine were mixed with wild-type cells in reconstruction experiments. The colony-forming ability of the resistant cells was measured under various selective conditions. Choice of antimetabolite, size of resistant cell clusters, and overall plating density affect the efficiency of recovery of resistant colonies. The implications of these effects are discussed with respect to the isolation of mutants, the estimation of mutant frequency, and the importance of post-mutagenesis .treatment. The azauracil'-resistance phenotype should be useful as an indicator of mutagenesis since single resistant cells quantitatively produce visible colonies under selective conditions, and more than 2 × 106 total cells can be screened per plate.

Cultured plant~ell systems offer new possibilities for genetic manipulation of higher plants. Realization of the potential for genetic engineering of plants in culture will require the ability to produce mutant cell strains with specific phenotypic properties that are designed to fit specific needs. In animal cell systems, a careful evaluation of the parameters affecting the processes of mutagenesis and mutant selection has facilitated the production of mutant strains. (Chu, 1971; Fox, 1975). The growth characteristics of cultured plant cells, including cell clustering and density
92 Bourgin, 1978). Unfortunately, several of the techniques have serious shortcomings. Gradual enrichment selections (Bright and Northcote, 1975; Weber and Lark, 1979) and liquid-suspension selection procedures (Widholm, 1972; Gathercole and Street, 1978) do not permit accurate measurement of the frequency of resistant cells in a population. Furthermore, the nature of the variants isolated by such procedures is not immediately clear. Gradual enrichments may result in the accumulation of several small increments of resistance, while resistant suspensions may be a mixture of many cell lines. Ideally, one would like to apply microbial plating techniques to cultured plant cells so that mutants could be selected in a single step and individual resistant lines would remain spatially separated. Resistance phenotypes have been extensively used in animal cell studies as an indicator of m u t a t i o n induction. Such evaluation of mutagenesis requires the ability to monitor mutant frequency in control and treated populations. It is therefore important to be able to efficiently recover colonies from any cells with the selected phenotype that are present in a population. The characteristic growth of plant cells in clusters and the requirement of a minimum ceil density for growth may complicate the selection of mutants from plant cells (see Weber and Lark, 1979). In this report we describe experiments in which resistant cells are mixed with a large excess of wild-type cells and then are selected from this reconstructed population under various conditions. We show that choice of toxic agent, size of resistant cell clusters, and the density of cells on selective media affect the efficiency of colony formation by the resistant cells in a_population. Materials and methods

Plant material. H. Gracilis cell strain SR17-1 was initiated and maintained in Erikssons's medium (Eriksson, 1965) as previously described (Horsch and Jones, 1980). All suspension cultures were inoculated with 1 g fresh weight of cells per 100 ml medium in 500-ml erlenmeyer flasks and shaken at 140 rpm. Every 7 days cells were sieved through a Teflon screen (350-/~m mesh opening), collected on a Miracloth filter, and used to inoculate new suspension cultures. All cultures were grown at 28°C in the dark. The isolation and characterization of strain AG7 (resistant to 8-azaguanine [AG, Sigma]) were reported elsewhere (Horsch and Jones, 1978). Cells of H. gracilis grow in suspension in units ranging from single cells to clusters of more than a thousand cells. Cell clusters smaller than a given size were isolated from suspension cultures by filtration through Teflon screens of known mesh openings. Cell clusters larger than a given size were obtained by exhaustively washing smaller clusters through the proper size screen. Table 2 presents a typical distribution of clusters in a suspension culture of SR17-1 that passed through or were retained on different size screens. The 74-/~m filtrate typically contained 30--40% single cells. Strains resistant to the metabolite analogs 6-azauracil (AU, Sigma) or 5-hydroxylysine (DHL, Sigma) were isolated from cells of strain SR17-1 in the following way. Exponentially growing cultures were exposed for various periods to 1 of 3 mutagens: ethyl methanesulfonate (EMS; Sigma), N-methyl-

93 N'-nitro-N-nitrosoguanidine ( M N N G ; Sigma), or ultraviolet light (UV; Sylvania G 1 5 T 8 germicidal lamp). Details of the effects of these mutagens will be reported elsewhere (manuscript in preparation). Mutagenized cultures were allowed to recover and resume growth (about 3 culture mass doublings) before plating onto medium containing either I m M A U or 0.1 m M DHL. After 4--5 weeks colonies that grew on selective medium were transferred to normal growth medium and then retested for the ability to grow on selective medium. The resistant strains were maintained on or in normal medium and periodically retested for their ability to grow on medium containing the same analog at the same concentration. Details of the origin of each strain are given in Table 1. Some of the AU-resistant strains were characterized in more detail in another report (Jones and Hann, 1979). Filter paper growth assay. Growth of cells on normal and AU- or DHL-containing medium was measured using the filter-paper growth assay technique (manuscript in preparation). Suspension cells in exponential growth (3--5 days after transfer) were filtered through a Teflon screen (350-~m mesh opening), collected on a Mimcloth filter, and resuspended in liquid medium at a density of 0.1 g fresh weight of cells per mh Aliquots o f 0.5 ml were pipetted onto the surfaces of 7 ~ m discs of Whatman No. 2 (qualitative) filter paper resting on the surface of agar~olidified (0.8% w/v) medium in 1 0 0 × 15 mm plates. After the excess liquid had absorbed into the agar, each plate was weighed with and without the filter paper and cells. The difference between these weights was the weight of the filter paper and the inoculum; by subtracting the weight of the inoculum (0.05 g) it was possible to calculate the tare weight of the filter paper alone (including the weight of medium it adsorbed from the agar). At daily intervals each plate was weighed again with and without the filter paper and cells. The filter-paper tare weight was subtracted from each value to give the weight of the growing ceils. Variation between replicate assay plates was small; standard errors usually were much less than 10% of the means. TABLE 1 ORIGIN OF VARIANT STRAINS

Strain

Parent

Mutegen a

Cloned b

AUH2 AUH4 AU1

SR17-1 SR17-1 AG7

UV UV EMS

Yes ~" No ~ No

AU3 AU4 AU5

AG7 AG7 AG7

EMS EMS EMS

DHLI DHL2 DHL4

S R I 7-1 SR17-1 SR17-1

UV UV MNNG

DHL5

SR17-1 S R I 7-1

UV MNNG EMS

DHL6 DHI 7

SR 17-1

No. No • -No Yes ' Yes

Yes Yes No Yes

a Parental cultttres were treated w i t h the indicated m u t e g e n b e f o r e s e l e c t / o n . b S o m e o f t h e variant strains were c l o n e d us/rig the d o u b l e fi/ter--paper plating t e c h n i q u e (Horsch and Jones, 19S0).

94 TABLE 2 D I S T R I B U T I O N OF C E L L C L U S T E R S IN A T Y P I C A L 7-DAY OLD SUSPENSION C U L T U R E Size o f c l u s t e r s a (/~m)

~74 74--177 177--350 350--420 420--500 ~500

Cells p e r c l u s t e r

P r o p o r t i o n o f t o t a l c u l t u r e (% b y weight)

Range

Average

1-5 5-- 35 35--120 120--300 300--500 --

2 15 60 180 ---

3 39 34 7 7 10

a T h e c u l t u r e was i n i t i a t e d b y i n o c u l a t i n g 1 . 0 g f r e s h w e i g h t o f cells f r o m a 3 5 0 Dm f i l t r a t e i n t o 1 0 0 m l o f m e d i u m . A f t e r 7 d a y s , t h e clusters in t h e c u l t u r e w e r e s e p a r a t e d i n t o d i f f e r e n t size r a n g e s b y e x h a u s t t i v e l y sieving c l u s t e r s t h r o u g h o r c o l l e c t i n g t h e m o n T e f l o n s c r e e n s w i t h t h e i n d i c a t e d m e s h o p e n i n g s .

Reconstructions. Wild-type and AU- or DHL-resistant strains were mixed together in various combinations and plated onto selective medium to determine the efficiency of colony formation from the resistant cells. The selective medium contained 1 mM AU or 0.1 mM DHL, depending upon the p h e n o t y p e of the resistant strain being tested. Wild-type cells were filtered through a 350-/~m mesh opening Teflon screen before use. Results

Characterization o f cell strains DHL1 and A U H 2 are typical of the variant strains isolated from SR17-1. A comparison of their growth in the presence o f D H L or AU with that of SR17-1 is given in Figs. 1 and 2. In each case, SR17-1 was unable to continue growth at concentrations of the analogs that had little or no effect on the growth of the corresponding resistant strain. Growth was monitored for an additional 2 weeks b e y o n d the initial 15 days reported in Figs. 1 and 2. SR17-1 continued to grow slowly on medium containing 0.01 mM DHL, b u t completely ceased to grow after an initial doubling of fresh weight on medium containing 0.03 mM D H L (data n o t shown). SR17-1 continued to grow slowly on medium containing 0.03 mM AU b u t n o t on the higher concentrations .tested. To test the reliability o f the filter-paper growth assay, the response of SR17-1 and A U H 2 to AU was measured in suspension culture. Similar results were ~btained (data n o t presented). Cultures of SR17-1 and most AU- and DHL-resistant strains grew with comparable vigour and color in suspension and on solid media. There was no apparent difference in the degree o f aggregate dispersion between SR17-1 and most variant strains. DHL- and AU-resistant strains exhibited normal sensitivity to AU and DHL, resp. All variant strains used in this study have maintained their resistance phenotype(s) for long periods in the absence of selection and were c o m p o s e d primarily of diploid cells (typically 95% diploid with the remainder being tetraploid). Strain AG7 has remained resistant to 6.6 #M AG for more than 3 years of continuous suspension culture in the absence of AG. Strains AU1, AU3,

95

~o

IO r-1

/

1

//°

:

i' ~1.

o.,/; r

O

5 ~ ~ROWTH TIME (doys)

O

• • I ~ = IO GROWTH TIME (days)

•

Fig. 1. G r o w t h o f S R 1 7 - 1 a n d D H L 1 i n m e d i u m cont.~tntng v a r i o u s c o n c e n t r a t i o n s o f D H L . F r e s h w e i g h t s o f d u p l i c a t e c u l t u r e s w e r e m e a s u r e d d a i l y u s i n g t h e f i l t e r p a p e r g r o w t h a a a s y . S R 1 7 - 1 cells i n m e d i u m w i t h n o d r u g o r w i t h 0 . 0 0 2 m M D H L ( e ) ; S R 1 7 - 1 eell~ i n m e d i u m w i t h 0 . 0 0 4 m M D H L (m); S R 1 7 - 1 c e n s in m e d i u m w i t h 0 . 0 1 m M D H L (&); D H L 1 cells i n m e d i u m w i t h n o dlmg o r w i t h 0 . 0 3 m M D H L (o); D H L 1 cells in m e d i u m w i t h 0 . 1 m M D H L ( v ) ; D H L 1 eeli~ i n m e d i u m w i t h 0 . 3 m M D H L (4). Fig. 2. G r o w t h o f S R 1 7 - 1 a n d A U H 2 i n m e d i u m c o n t a i n i n g v a r i o u s c o n c e n t r a t i o n s o f A U . F r e s h w e i g h t s o f d u p l i c a t e c u l t u r e s w e r e m e a s u r e d d a i l y u~in_~ t h e f i l t e r p a p e r g r o w t h a s s a y . S R 1 7 - 1 cells i n m e d i u m w i t h n o d r u g ( e ) ; 8 R 1 7 - 1 eelis i n m e d i u m w i t h 0 . 0 3 m M A U (#); S R 1 7 - 1 eeli~ in m e d i u m w i t h 0 . 1 m M A U ( s ) ; S R 1 7 - 1 cells i n m e d i u m w i t h 0 . 3 m M A U (A)i A U H 2 c e l l s i n m e d i u m w i t h n o d r u g o r w i t h 0 . 3 m M A U ( o ) ; A U H 2 cells i n m e d i u m w i t h 1 . 0 m M A U (~).

AU4 and AU5 (which were derived from AG7) were resistant to both 1 mM AU and 6.6 #M AG. Reconstructions To evaluate the efficiency of variant recovery under selective conditions, populations of cells were constructed by mixing a few variant cells with a large n u m b e r of wild-type cells. The proportion of variant colonies recovered from these populations was determined under various selective conditions. We examined the effects of 2 parameters on the recovery of m u t a n t colonies from variant cells that had been mixed with wild-type cells. These parameters were the initial size of the variant cell clusters, and the initial density of cells on selective plates. The results of an experiment in which D H L 5 cells were mixed with an excess of A U H 2 cells and then plated onto medium containing 0.1 mM D H L are presented in Table 3. These results demonstrate that b o t h the initial size of variant cell clusters and the initial p h t i n g density can strongly influence the recovery o f DHL-resistant colonies. DHL-reslstant cells must be pr~esent in clusters o f 35 cells or more before they can grow under these selective conditions. Furthermore, at densities above 2 X l 0 s cells per plate, the efficiency of recovery of resistant clusters drops significantly. These constraints necessitate

96 TABLE 3 DHL RECONSTRUCTION: PERCENTAGE OF DHL5 CLUSTERS COLONIES UNDER VARIOUS SELECTIVE CONDITIONS N u m b e r of D H L 5 cells p e r c l u s t e r

no D H L 5 I-- 35 35--120 120--500

cells b c d

WHICH

PRODUCED

VISIBLE

R a t i o o f visible c o l o n i e s p r o d u c e d t o c l u s t e r s o f r e s i s t a n t cells s e e d e d (%) a 2 × 105 t o t a l cells/plate

5 × 10 $ total cells/plate

0
0 1 28 36

1 × 106 t o t a l cells/plate 0 <1 1 9

a C o l o n i e s w e r e c o u n t e d 3 w e e k s a f t e r p l a t i n g o n m e d i u m c o n t a i n i n g 0.1 m M D H L . P e r c e n t a g e s are t h e a v e r a g e o f c o l o n i e s p r o d u c e d o n 5 plates. A U H 2 cells w e r e u s e d as t h e w i l d - t y p e ( D H L - s e n s i t i v e ) in this experiment. b 60 D H L 5 clusters were seeded per plate. c 21 D H L 5 c l u s t e r s w e r e s e e d e d p e r p l a t e . d 5 D H L 5 clusters were seeded per plate.

the use of long recovery periods after mutagenesis and require large numbers of selective plates to screen a population for DHL-resistant cells. As a control on the origin of DHL-resistant colonies t h a t developed on the selective plates in this experiment, AUH2 cells were used as the DHL-sensitive cells. All of the DHL-resistant colonies were tested for ability to grow on medium containing 1 mM AU: they all were completely inhibited. Thus all of the DHL-resistant colonies were derived from the DHL5 cells t h a t were seeded into the AUH2 cells. In addition, this result strongly suggests that few or no viable AUH2 cells were transferred from the original selection plates to the AU-conraining medium. The results of an AU reconstruction experiment are shown in Table 4. In this experiment AUH2 cells were mixed with an excess of DHL1 cells plated o n t o medium containing 1.0 mM AU. Even single resistant cells or small clusters o f 2--5 resistant cells were able to produce colonies under selective conditions. More than 2 × l 0 s cells could be screened per plate w i t h o u t reducing the efficiency of recovery of resistant cells. Any new, spontaneous AU-resistant colonies arising from the DHL1 cells should have been able to grow on 0.1 mM DHL. The AU-resistant colonies all were scored for ability to grow in the presence of 0.1 mM D H L : ' t h e y all were inhibited. Thus all of the colonies developing in this reconstruction experiment arose from the pre-existing variant cells seeded into the DHL1 population. In a reconstruction experiment using the doubly marked strain AU3, some of the AU-resistant colonies t h a t developed were scored for the AG-resistance phenotype. 49 o u t of 50 colonies tested were able to grow on 6.6/~M AG. The frequency o f spontaneous AU- or DHL-resistant cell clusters in strain SR17-1 ranges from 0.2 to 1 × 10 -~ per colony-forming unit. This is sufficiently low t h a t spontaneous resistant colonies should only rarely occur in a reconstruction experiment. In cases where the spontaneous frequency is high, the use of multiply or differently marked strains would allow one to distinguish colonies arising from the seeded resistant cells from those occurring spontaneously.

97 TABLE 4 AU RECONSTRUCTION: PERCENTAGE OF AUH2 CLUSTERS COLONIES UNDER VARIOUS SELECTIVE CONDITIONS Number of AUH2 cel/s p e r c l u s t e r

n o A U H 2 cells 1--5

WHICH

PRODUCED

VISIBLE

R a t i o o f visible c o l o n i e s p r o d u c e d t o c l u s t e r s o f r e s i s t a n t eel/s s e e d e d (%) a 5 X 10 ~ t o t a l cells/plate

1 X 10 6 t o t a l cells/plate

2 X 10 6 t o t a l cells/plate

4 X 10 ~ total cells/plate

0 88

0 106

0 118

0 71

a C o l o n i e s w e r e c o u n t e d 4 w e e k s a f t e r plating o n m e d i u m c o n t a | n ! n ~ 1 . 0 m M A U . P e r c e n t a g e s are t h e average o f c o l o n i e s p r o d u c e d o n 5 p l a t e s . D H L 1 cells w e r e u s e d as t h e w i l d - t y p e ( A U - s e n s t , t i v e ) i n this Expt. 17 AUH~ clusters were seeded per plate.

To establish the generality of these results, reconstructions were performed with several independently isolated strains of each phenotype. The data in Tables 5 and 6 demonstrate that the response of DHLo and AU-resistant cells to the different selection parameters is typical of each phenotype and not just a property o f one specific strain. . We previously reported the isolation of AG-reslstant strains (Horsch and Jones, 1978). Subsequent experiments have revealed that colonies with this phenotype can only be selected under conditions similar to those reported for the isolation o f AGT: reconstructions with AG7 and SR17-1 on medium containing 6.6 #M AG under conditions similar to those described for DHL or AU reconstructions failed to demonstrate any combination of factors where AG7 cells could produce colonies. AG7 cells could produce colonies only under very mild selective conditions that permitted development of many non-resistant colonies as well (data not presented). TABLE 5 DHL RECONSTRUCTION: PERCENTAGE OF DHL'RESISTANT CLUSTERS VISIBLE COLONIES UNDER VARIOUS SELECTIVE CONDITIONS DHL-resistant strain

R a t i o o f v/sible c o l o n i e s p r o d u c e d t o c l u s t e r s o f r e s i s t a n t cells s e e d e d (%) a

8 X I0 $ total

DHL1 DHL2 DHL4 DHL5 DHL6 DHL'~

WHICH PRODUCED

2.4 X 106 total

1 - - 3 5 cells per cluster b

3 5 - - 1 2 0 cells per cluster c

3 5 - - 1 2 0 cells per cluster c

3 ~I ~I ~1 ~1 ~1

94 16 S9 89 90 28

~1 ~1 5 16 5 ~1

a C o l o n i e s w e r e c o u n t e d 4 w e e k s a f t e r platin~ o n m e d i u m c o n t e i n i n ~ 1 . 0 m M D H L . P e r c e n t a g e s ere t h e average o f c o l o n i e s p r o d u c e d o n 5 p l a t e s . S R 1 7 - 1 cells w e r e u s e d as t h e .wild-type in this E x p t , b 12 DHL-rsetstent clusters were seeded per plate. c S DHL-rseistant clusters were seeded per plate.

98 TABLE 6 AU RECONSTRUCTION: PERCENTAGE OF AU-RESISTANT CLUSTERS VISIBLE COLONIES UNDER VARIOUS SELECTIVE CONDITIONS

WHICH PRODUCED

AU-resistant strain

R a t i o o f visible c o l o n i e s p r o d u c e d t o clusters o f r e s i s t a n t cells s e e d e d (%) a

~UH2 AUH4 AU1 AU3 AU4 AU5

91 95 96 96 89 75

a C o l o n i e s w e r e c o u n t e d 4 w e e k s a f t e r p l a t i n g o n m e d i u m c o n t a i n i n g 1.0 m M A U . P e r c e n t a g e s are t h e a v e r a g e o f c o l o n i e s p r o d u c e d o n 5 p l a t e s . T h e r e w e r e 2 X 106 t o t a l cells p e r p l a t e a n d S R 1 7 - 1 cells w e r e u s e d as t h e w i l d - t y p e . A b o u t 6 0 A U - r e s i s t a n t ceils a n d c l u s t e r s f r o m a 74-~rn f i l t r a t e w e r e s e e d e d p e r plate.

Discussion SR17-1 and AU-, DHL-, and AG-resistant strains can be easily and unequivocally identified b y a simple test of their ability to grow on medium containing 1.0 m M AU or 0.1 mM D H L or 6.6/~M AG. This p r o p e r t y coupled with the vigorous growth capacity of the variant strains makes possible mixed population studies such as those reported here or elsewhere (Horsch and Jones, 1980). Since cultured cells of H. gracilis can n o t y e t b e induced to regenerate whole plants, the genetic nature of our variants has n o t been established. As we have discussed in previous reports (Horsch and Jones, 1978; Jones and Harm, 1979), these variant strains m e e t certain criteria which suggest that they are mutants: (1) the variants were isolated in a single selective step; (2) the frequency of such variants ( ~ 1 0 -6 ) is in the range expected of genetic variants; (3) their p h e n o t y p e s are stable for long periods in the absence of selection; and (4) in the case of AU-resistant vat~iants, different strains exhibit a different spectrum of cross resistances to related analogues (Jones and Harm, 1979). While determination of the genetic status of variants is important to many studies, it is probably of secondary importance to this report since it is the phenotypic properties rather than the genotype of the variants that directly influences their colony-forming ability under selective conditions. Reconstruction experiments such as we have described here permit evaluation of several parameters affecting selection of colonies with variant phenotypes. The fact that our cultures were affected differently by AG, D H L and AU demonstrate the necessity of testing each selective agent to be used. There is a possibility that certain selective schemes would never permit recovery of cells with the desired p h e n o t y p e . One parameter of importance is the minimum n u m b e r of variant cells that must be present in a cluster for efficient growth under selective conditions. We have demonstrated that, with H. gracilis, DHL-resistant cells cannot be isolated from an excess of DHL-sensitive cells unless the resistant cells are present in clusters of 35 cells or more. In an earlier publication we demonstrated that cells and clusters in a 74-#m filtrate of DHL1 could grow efficiently under non-

99

selective conditions using our double filter paper plating system (Horsch and Jones, 1980). Thus the poor recovery of colonies from DHL-resistant clusters with less than 35 cells is not due to some inherent property of the DHL-resistance phenotype. It is possible that DHL-inhibited wild-type cells are antagonistic to the growth of small clusters of DHL-resistant cells. A phenomenon such as this necessitates a re~valuation of several aspects of mutant induction and isolation. The period of recovery following mutagenesis must allow sufficient time for segregation of mutational damage and growth of any new variant cells to clusters large enough for efficient recovery under selective conditions. If cultures are subjected to selection before these events occur, the induced mutations will be lost; only preexisting mutants in sufficiently large clusters (if any) will be recovered. Another parameter that can influence the efficiency of mutant recovery is the overall density of cells plated onto selective medium. Because of the problem of density~lependent growth, when too few cells are present under any conditions, colony formation can not occur. When a large number of cells is present under selective conditions, metabolic cooperation such as cross-feeding or excretion of toxic compounds by dying cells might interfere with growth of resistant cells. Such a "'cooperative death" syndrome has been suggested (Weber and Lark, 1979; Chu and Lark, 1976). The efficiency of recovery of DHL-resistant cells is markedly reduced by higher densities of wild-type cells, while the efficiency of recovery of AUresistant cells is much less affected by total cell density. The practical implication of this density phenomenon is that there are limits on the number of cells that can be screened for variants in a given volume of medium. One possible difficulty cannot be tested by a reconstruction experiment: variant cells present in a mixed cluster (mutant and wild-type cells in the same cluster) might not grow under selective conditions because of metabolic interaction with the wild-type cells present in the cluster. If this is the case, the recovery period would have to be adjusted to permit dissociation of wild-type and variant cells and growth of pure variant colonies to at least the minimum size for selection. A complication arising from working with cell clusters involves their effect on determination of mutant frequency. If a culture consisted entirely of single cells, then each variant cell would be expected to produce one variant colony under selective conditions. Mutant frequency would be the ratio of the number of variant colonies to the total number of cells tested. However, since plant~ell cultures generally consist of a mixture of single cells and clusters of a few cells to several hundred cells, mutant frequency cannot be easily determined on a "per cell" basis. Since one actually measures the number of colonies that are produced by cell clusters, frequencies should be reported on a "per colonyforming unit" basis. We have demonstrated that a reasonable estimate of mutant frequency can be made only after careful evaluation of the selective scheme used. It should be noted that a resistant strain is required for testing the selective system. Therefore the inability to isolate a certain mutant cannot be used to imply that such mutants are not present in the population screened. We have evaluated the efficiency of colony formation by resistant cells on

100

medium containing AG, DHL or AU. Only AU-resistant cells can quantitatively produce colonies from single cells or small cell clusters in the presence of very large numbers of wild-type cells. Since the frequency of AU-resistant cells and clusters can be accurately and easily measured, this phenotype should be useful for assessing the effectiveness of mutagenesis on cultures of H. gracilis (manuscript in preparation). Acknowledgements We thank D. Cooksey, J. Hann and C. Ward for excellent technical assistance. This work was supported by grant number PCM75-21779 from the National Science Foundation and by funds from the Agricultural Experiment Station, University of California, Riverside. References Bottrgin, J.P. (1978) I s o l e m e n t de m u t a n t s b pa~ttr de cellules v~g~tales en culture in vitro, Physiol. Veg., 16, 339--351. Bright, S.W.J., and D.H. N o r t h c o t e (1975) A deficiency of h y p o x a n t h i n e phosphoribosyltransferase in a sycamore callus resistant to azaguanine, Planta, 123, 79--89. Chu, E:H.Y. (1971) I n d u c t i o n and analysis of gene m u t a t i o n s in m a m m a l i a n cells in culture, in: A. Hol.laender (Ed.), Chemical Mutagens, Principles and Methods for Their Detection. Vol. 2. Plenum, New York, pp. 411--444. Chu, Y., and K.G. Lark (1976) Cell cycle parameters of s o y b e a n (Glycine max L.) cells growing in suspension culture: Suitability of the system for genetics studies, Plants, 132, 259--268. Erlksson, T. (1965) Studies on the growth r e q u i r e m e n t s of~cell eultttres of HaPloP,apPus g~acitis, Physiol., Plant., 18, 976--993. Fox, M. (1975) Facto rs affecting the q u a n t i t a t i o n of dose--response curves for m u t a t i o n i n d u c t i o n in V79 Chinese h a m s t e r cells after exposure to chemical and physical mutagens, M u t a t i o n Res., 2 9 , 4 4 9 - - 4 6 6 . Gathereole, R.W.E., and H.E. Street (1978) A p-fluorophenylalanine-resistant cell line of sycamore w i t h increased c o n t e n t s of phenylalanine, tyrosine and phenolics, Z. Pflanzenphysiol., 89, 283--287. Horsch, R.B., and G.E. Jones (1978) 8-Azaguanine-resistant variants of cultured cells of Haplopappus gracllis, Can. J. Bot., 56, 2660--2665. Horseh, R.B., and G.E. Jones (1980) A double filter paper t e c hni que for plating cultured pl a nt cells, In Vitro, 1 6 , 1 0 3 - - 1 0 8 . Jones, G.E., and J. Harm (1979) H~plopappus gracilis cell strains resistant to pyrim~dine analogues, Theor. AppL Genet., 54, 81--87. Maliga, P. ( ! 9 7 8 ) Resistance m u t a n t s and their use in genetic m a n i p u l a t i o n s , in T.A. Thorpe (Ed.), Frontiers o! Plant Tissue Culture 1978. Int. Assoc. for Plant Tissue Culture, Calgary, Alberta, Canada, pp. 382--392. Weber, G., and K.G. Lark (1979) An efficient plating system for rapid isolation of m u t a n t s ~ o m pl a nt cell suspensions, Theor. AppL Genet., 55, 81--86. Widhohn, J.M. (1972) A n t h r a n l l a t e s y n t h e t a s e from 5-methyltaTptophan-suseeptible and -resistant cultured Daucus carota cells, Binchim. Biophys. Acta, 279, 48--57. Widhohn, J.M. (1 977) Selection and characterization of bi oc he mi c a l m u t a n t s , in: W. Barz, E. Ralnhard and M.H. Zenk (Eds.), P h n t Tissue Culture and its Bin-Technological Application, Springer, New York, pp. 112--122.