Plant Cell Reports (1985) 4:151-154 Plant Cell Reports © Springer-Verlag 1985 Phenylalanine and tyrosine accumulating cell lines of a dihaploid potato selected by resistance to 5-methyltryptophan E. Jacobsen, R. G. F. Visser, and J. Wijbrandi Department of Genetics, Centre of Biological Sciences, Kerklaan 30, University of Groningen, Groningen, ]]a.e Netherlands Received November 26, 1984 / Revised version received April 11, 1985 - Communicated by P. Maliga Abstract The selection and characterization of 5-methyltrypto- phan (5-MT) resistant cell lines is described in a dihaploid potato, clone H2578(007). The frequency of resistant calli was increased by treatment of a cell suspension with N-ethyl-N-nitroso-urea. Two lines, 5-MT-2| and 5-MT-26, accumulated tyrosine (160 nMol and |546 nMol/g fresh callus), and the third, 5-MT-27, accumulated both tyrosine (84] ~Mol/g fresh callus) and phenylalanine (45| nMol/g fresh callus). In the wildtype tyrosine and phenylalanine content was 65 nMol and 42 ~Mol/g callus, respectively. The trypto- phan content of line 5-MT-26 was significantly in- creased, from 20 nMol/g to 76 nMol/g fresh callus. The total free amino acid content of the three vari- ant cell lines wss higher than that of the wildtype. Variant cell lines 5-MT-21, -26 and -27 showed a low degree of resistance to 5-MT, when grown on a selec- tive medium and were cross-resistant to parafluoro- phenylalanine and 3-fluorotyrosine, analogues of phenylalanine and tyrosine. Abbreviations: ENU, N-ethyl-N-nitroso-urea; 5-MT, 5-methyltryptophan; 3-FT, 3-fluorotyrosine; PFP, parafluorophenylalanine; AEC, S-(2-amino-ethyl) L- cysteine. Introduction Isolation of cell lines resistant to amino acid ana- logues (reviewed in Maliga |980) such as 5-MT have been described in carrot (Widholm 1972a and |974), Datura (Ranch et al. ]983) and tobacco (Widholm 1977). 5-MT resistant cell lines were also obtained in a tetraploid potato variety (Carlson and Widholm |978). The growth of these cell lines was highly resistant to the analogue, the cells accumulated tryptophan and possessed an altered anthranilate synthase. In the present paper, the induction and isolation of cell lines of a dihaploid potato are described which were selected by the ability to grow on a selective 5-MT medium. Some of the cell lines were found to accumulate phenylalanine and/or tyrosine. These cell lines have been tested for 5-MT resistance and cross resistance with analogues of tyrosine and phenyl- alanine. Material and Methods Plant material: After surface sterilizatio~ of young leaves (Jacobsen ]98|) of a dihaploid potato (clone H 2 578 (=007), kindly supplied by Dr. G. Wenzel, Offprint requests to: E.Jacobsen Institut f~r Resistenzgenetik, Gr~nbach, BRD), callus was induced on cut leaf pieces and subcultured on basal Murashige and Skoog (|962) (MS) medium supple- mented with 5 mg/l s-naphthalene-acetic acid (NAA) and 0.| mg/l benzyl amino purine (BA~ at 25°C in the dark. Cell suspensions were made by suspending callus through a sieve (670 ]Jm) and growing the cells in liquid medium containing the same salts, NAA and BAP concentrations as above, on a rotary shaker (|20 rpm) at 25°C in the dark. Growth assay: The inhibiting effect of 5-MT, 3-FT or PFP, on callus growth was determined by transferring three small pieces (5-|0 mg each) of callus onto me- dium containing different concentrations of one of these analogues. After four weeks of growth the in- crease in fresh weight was determined and compared with that of callus grown on analogue-free medium. For each treatment and for control three Petri dishes (~ 5 cm) were used. Mutagenic treatment: Three days after subculturing, freshly sieved (670 ~m) cell suspensions, containing 2 x ]05 cells/ml, were treated with ]0, 50 or |00 ~M ENU. After four days of incubation with ENU, the treated cells were plated at a density of ] x |05 cells/ml in solid medium containing variou s concen- trations of 5-MT. The percentage of viable cells was determined by staining with phenosafranine (Widholm |972b). Per Petri dish (~ 9 cm), 2,5 ml of cell sus- pension and 2,5 ml of medium containing |0 g/l agar (Colijn et al. ]978) supplemented with 5-MT was added. - - _ _ The final 5-MT concentration was the same in the cell containing layer and in the plate. After thorough mixing of the plated cell suspension with agar con- taining medium, the plates were incubated in the dark at 25°C. Per ENU treatment and 5-MT concentration 4.]07 cells were plated. After 5-7 weeks, resistant calli were scored. Amino acid composition: A. Q~'a'litative determination: Callus extracts were prepared according to Bieleski and Turner (]966). Per cell line, ]00-200 D1 of extract was spotted on a thin-layer plate (0.| ~mn PC-cellulose). After thor- ough drying, the plate was placed in a chromatography chamber containing a mixture of n-butanol/acetic acid/ H20 (4:|:]) or ethyl alcohol/H20 (7:3). After ap- proximately 5 h the plate was removed from the cham- ber, dried and sprayed with ninhydrin reagent (0.25% ninhydrin in 95% ethyl alcohol). The plate was heated for | h at 60°C, in some cases followed by a specific staining for tryptophan by the Ehrlich reaction