Partial characterization of two stable auxotrophic cell strains of Datura innoxia Mill

Planta 149, 480484(5980) P I ~ H ' ~

�9 by Springer-Verlag 1980

Partial Characterization of Two Stable Auxotrophic Cell Strains of Datura innoxia Mill.

John King, R o b e r t B, Hor sch , and A r t h u r D. Savage

Department of Biology, University of Saskatchewan, Saskatoon, Sask. S7N 0W0, Canada

Abstract. A growth analysis o f several p re sumpt ive " l e a k y " a u x o t r o p h s f rom D a t u r a i n n o x i a suspens ion cul tures led to the d iscovery of an aden ine - requ i r ing cell s t ra in (Ad 1). Both A d 1 and Pn 1, a pan to the na t e - requi r ing s t ra in i so la ted ear l ier f rom these cultures, still requi re e i ther adenine or p a n t o t h e n a t e for g rowth after m o r e than one year in culture. A t t e m p t s to select p r o t o t r o p h i c rever tan ts have failed. A d 1 also grew well in a m e d i u m con ta in ing ei ther 5 -amino imidazo le - 4 - c a r b o x a m i d e r ibo t ide or inos ine ins tead o f aden ine ; Pn t with pan to i c acid a lone b u t n o t at all in the presence o f / L a l a n i n e or c~-ke to i sovalera te a lone in- s tead of pan to thena te . Pn 1 cells s ta rved of pan to the n - ate for up to 4 d and A d l o f adenine for 10d or m o r e r e sumed g rowth when t rans fe r red to a p p r o p r i - a tely supp l emen ted media . Wi ld - type D a t u r a cells g rown on u n s u p p l e m e n t e d m e d i u m wou ld no t cross- feed the requi red nut r ien ts to the auxo t rophs . The s t a rva t ion and cross- feeding exper iments showed tha t

b o t h a u x o t r o p h s cou ld be used in r econs t ruc t ion exper iments to deve lop enr ichment - se lec t ion techniques for the i so la t ion of m o r e auxo t rophs .

Key words: Aden ine - A u x o t r o p h - Cell cultures, va r i an t lines - D a t u r a - Pan to thena te .

Introduction

In a previous repor t , we discussed the ear l ier a t t empts by others to isolate a u x o t r o p h s f rom p lan t tissue cultures and descr ibed ou r non-select ive m e t h o d s for the i so la t ion o f a u x o t r o p h s f rom p r e d o m i n a n t l y hap- lo id D a t u r a i n n o x i a cell cul tures [12]. Th roug h the use of these me thods a cell s t ra in (Pn 1) was i so la ted tha t had an abso lu te r equ i remen t for p a n t o t h e n a t e in o rde r to grow [12]. F r o m a m o n g the 2,370 colonies which y ie lded Pn 1, several p r e s u m p t i v e " l e a k y " auxo-

t rophs were also ident i f ied one of which (Ad 1) was shown, after fu r ther testing, to have an abso lu te requ i r emen t for adenine in o rde r to grow. In the present paper , add i t i ona l character is t ics o f Pn 1 and a descr ip- t ion of A d 1 are given, as well as an assessment of the po ten t i a l usefulness o f bo th a uxo t rophs in recon- s t ruc t ion exper iments des igned to test poss ible enr ichment -se lec t ion p rocedures for a u x o t r o p h recovery.

Materials and Methods

Cell Strains; Culture. The origin of the predominantly haploid cultured cell strain, DI-6, of Datura innoxia (Mill.) used as source material in these experiments has been reported [3, 52]. DI-6, and a fast-growing strain, Ph 5, derived from DI-6, were grown in B 5 medium [4] modified by adding 0.5 g. 1-1 NH4NO3 and 1 rag. 1-1 of 2,4-dichlorophenoxyacetic acid (BsA medium). The Adl and Pn5 strains were routinely maintained in B~A medium supplemented with 67.5 mg- 1 - 1 of adenine or 2 rag. 1 1 of Ca-D-pantothenate, respectively. When solid media were used, 0.8% (w/v) of agar was added to liquid.

Stock cultures were maintained by inoculating 10 mI of cell suspension into 50 ml of the appropriate medium contained in 250 ml Delong (BellCo, Vineland, N.J., USA) flasks, at 5-d intervals. Calli growing on solid media in Petri plates (500 mm diameter, 15 mm high) were routinely transferred at 14--25-d intervals.

All cultures were grown in continuous light (1.614 W m-2; from "cool white" fluorescent lamps, Westinghouse, Pittsburgh, Pa., USA) at 28 ~ C, the liquid cultures on a rotatory shaker (model G-10; New Brunswick Co., Edison, N.J., USA) at 150 rpm (2.5 cm ecc.)

Growth Analyses. The filter-paper growth assay (FPGA) [7] was used to determine the growth ofAd 5 (Figs. 1, 2) and in starvation experiments (Figs. 4, 5).

In growth experiments with Ad 5 (Figs. 5, 2), cal5i, grown on solid media for 7-14 d, were macerated in a sterile Petri plate and suspended in BfA medium. The suspension was passed through a 500-gin Teflon screen (Albert Godde Bedin Sales, Elmsford, N.Y., USA). The cells in the filtrate were collected on Miracloth (Chicopee Mills, New York, N.Y., USA) discs, allowed to drain, and then resuspended in BfA medium to a concentration of 0.2 g wet weight of cells ml- 1. Portions of this suspension, 0.5 ml each, were pipetted onto sterile, 7 cm discs of Whatman (Clifton, N.J.) No. 2 filter paper in Petri plates (100 mm diameter, 15 mm high)

0032-0935/80/0149/0480/$01.00

J. King et al.: Auxotrophic Cell Strains of Datura 481

containing 25 ml of solid B5A medium supplemented with appropriate metabolites. The plates were gently tilted and rotated to distribute the cells, and weighed under sterile conditions. The filter papers were then lifted from the plates with sterile forceps, the plates weighed again, and the filter papers replaced. At suitable intervals, the plates and the filter papers with the inocula were reweighed in the same way, the difference giving the wet weight of the growing culture.

In the starvation experiments (Figs. 4, 5), 7-d A d l or 4-d Pn 1 suspension cultures were filtered through 500-gm screens, collected on Miracloth filters as above, and washed with sterile distilled water. Filter-paper growth-assay plates containing B 5A medium were inoculated with either 0,2 g wet weight of Pn 1 cells or 0.1 g of Ad 1 cells per plate. At 2-d intervals triplicate cultures were transferred to Petri plates containing BsA medium supplemented with the required metabolite. The growth of the cells was followed using the FPGA technique.

Suspensions were used to obtain the results in Fig. 3. Twenty ml of a 3-d-old Pn l suspension were collected on a Miracloth disc, washed with BsA medium, and resuspended in 100 ml of the same medium. After a 4-d incubation, 10-ml portions of this culture were inoculated into 50 ml of BsA medium supplemented as in Fig. 3.

The dry weights of the cells were determined by removing two 5-ml samples from flasks at intervals. The cells were collected on Miracloth discs, washed with distilled water, and dried for 24 h at 60 ~ C.

Revertant Selection. Cultures of A d l or Pn 1 grown in standard liquid media for 7d were screened and resuspended as were Ad 1 cells in the growth experiments (see Growth Analyses above). Por- tions of cell suspension, 1 ml each, were spread evenly onto solid BsA medium in 10 (for Pn l ) or 20 (for A d l ) Petri plates (as above). Control plates contained BsA medium supplemented with either 67.5 m g . l - 1 of adenine or 2 mg.1-1 of pantothenate. The plates were incubated for either 30d (Pnl) or 42d (Adl) and then inspected for growing colonies. In the case of A d l , about 5.105 colony forming units representing about 8.106 cells were screened, and for Pn 1, about half that number.

Cross-Feeding. The double-filter-paper plating system [6] was used to determine whether nutrients which could satisfy the auxotrophic requirement of Ad 1 and Pn 1 could be supplied by wild-type cells placed adjacent to auxotrophic cells. Ph 1 cells were grown in BsA medium for 5 d, collected on a Miracloth disc, washed with distilled water, and 0.2 g wet weight of these "feeder cells" was spread evenly on the surface of medium in Petri plates (as above). Five replicate plates were used which contained BsA medium with or without the addition of 67.5 mg. l -~ of adenine or 2 mg-1 - I of pantothenate. The "feeder cells" were then overlaid by Whatman No. 2 filter papers each cut to fit exactly inside the rim of each plate. Ad 1 or Pn 1 cells, that had been grown in standard supplemented media for 5d, were passed through either a 500-~tm (Ad 1) or an 88-gm (Pnl) screen, and 0.5 ml portions of appropriately diluted filtrate pipetted onto 7-cm Whatman No. 2 filter papers resting on top of the larger filter papers covering the "feeder cells". These plates were then incubated for periods of up to 5 weeks.

Chemicals. Fine chemicals were purchased from Sigma Chemical Co., St. Louis, Mo., USA.

Results

The Growth of Ad l and Pnl. Figure 1 shows that in the absence of adenine, the wet weight of Ad 1

N 1,0

u

,~ 0.5

0.1

/ d ~ /~

. . . . . . .

0 . 0 5 i i i i ~ i i i i i i i i '/~ �9

0 5 10 21

Time (days) Fig. 1. The growth of the adenine-requiring auxotrophic cell line (Ad 1) of Datura innoxia with or without adenine. Cultures were grown on filter papers in Petri plates containing a medium without (o) or with adenine (mg . l -1 ) : �9 0.15; �9 0.6; ~ 3.0; [] 10; �9 67.5; �9 100

increased only slightly during a 21-d incubation period. Growth was progressively greater as the adenine concentration in the medium was increased from 0 to 67.5 mg. 1 - 1. When cells were grown at 100 mg. 1 - 1 of adenine, the increase in wet weight during a 10-d period was less than at 67.5 mg. 1-1

The growth of Pnl at increasing concentrations of pantothenate has been reported before [12]. Pn l did not grow in a medium supplemented with 0.25 mg. l -1 or less but grew nearly as well as the wild-type in a medium to which 2 mg. I - 1 of pantothenate was added [12].

In other experiments, the growth response of Ad 1 and Pn 1 in the presence of metabolites other than adenine and pantothenate was tested. The purine pre- cursor 5-aminoimidazole-4-carboxamide ribotide (AICAR) supported the continuous growth of Ad 1 after a lag of 4d when compared to adenine, as did inosine after a longer lag of 9d (Fig. 2). Inosinic acid supported very slow but measurable growth over a long period of time (results not shown).

The auxotrophic requirement for Pn 1 could be satisfied equally well by pantothenic and pantoic acids (Fig. 3) but less well by a mixture of pantoate and /%alanine. Neither ~-ketoisovalerate nor /~-alanine alone was an effective growth supplement.

Revertant Selection. All attempts to select prototrophic revertants from either strain were unsuccessful.

The Effect of Starvation on the Growth of Adl and Pnl. Figure 4 illustrates that A d l cells could be

482 J. King et al.: Auxotrophic Cell Strains of Datura

10-

5-

' ~ 0 . 5 -

..C

0,1-

e l m t,o .., ,oj.y o /o D /

l /= o7' 0 / I::]/

/:oo,/ eoO O . o ~ o - " �9

0.05~ i u u I i i i n t i n i n I u i i n I I I I I I I n

0 5 10 15 20 25

Time (days)

Fig. 2. The growth of the adenine-requiring auxotrophic cell line (Ad 1) of Datura innoxia in the presence of adenine or other purine nucleotide precursors. Cultures were grown as in Fig. 1 with 0.5 mM of: adenine (o), A!CAR ( - ) or inosine (D) �9 no supplement

1 2 -

' 8 E

E

O

5 4

O

I I

o 2 ; Time (days)

Fig. 3. The growth of the pantothenate-requiring auxotroph (Pn 1) of Datura innoxia in a liquid medium containing pantothenat e or suspected pantothenate precursors. �9 2 mg.1-1 Ca-D-pantothenate; �9 1.09rag.1-1 pantoic acid; �9 1.09mg.1-1 pantoic acid -t-0.75 rag.1 - I fl-alanine; z~ 1,16 rag- l - ' e-ketoisovalerate; o

0.75 mg.l ~ ]~-alanine; ~ no supplement

1~ t ' /

5

- L i / , /

"~ /},. ir ;0.1

'J o.,

t t t t t 0 05

Time (days)

Fig. 4. The growth of the adenine-requiring auxotroph (Ad 1) of D. innoxia following adenine starvation. Cells grown in standard supplemented medium for 7 d were filtered, washed and resuspended in unsupplemented medium. At 2-d intervals (indicated by ar- rows), portions of these starved cells were pipetted onto filter papers in Petri plates containing standard supplemented medium and growth measurements taken for several days. �9 No starvation. Starved for: o 2d; �9 4d; ~ 6d; �9 8d; A 10d

l O -

o

. / .P/"

//.< ... m'r

o

0 . 1 - i u u i u i u i n u l u u I , u 0 5 10 15 32

Time (days)

Fig. 5. The growth of the pantothenate-requiring auxotroph following pantothenate starvation. Cells were grown and starved as in Fig. 4. �9 No starvation. Starved for: o 2d; �9 4d; [] 6, 8 or 10d

starved of adenine for as much as 8 d without any significant change occurring in the rate at which cultures grew when transferred again to supplemented medium, except that the initial lag phase, before exponential growth ensued, increased slightly. Cells starved of adenine for 10d showed a pronounced lag

of about 4d before growing exponentially at a some- what slower rate than those cells starved for only 2-8 d (Fig. 4).

Pn 1 cells grew as well on supplemented medium after 2d of starvation as did unstarved cells (Fig. 5). After 4d of starvation a lag period of nearly 7d was

J. King et al. : Auxotrophic Cell Strains of Datura 483

seen before exponential growth occurred at a slower rate than for unstarved cells. Cells starved for 6d or more did not grow when transferred to supplemented medium. Cross-Feeding. When 160 Ad 1 units/plate or 200 Pn 1 units/plate were inoculated onto filter papers adjacent to "feeder cells" spread on B 5A medium (see Materi- als and Methods), no growth of the auxotrophs occurred over a 5-week period. In those Petri plates where the BsA medium was appropriately supplemented, an average of 132 or 140 Ad 1 or Pn 1 colonies were counted in each of the respective replicate plates.

Discussion

The results of the growth experiments with the Ad 1 cell strain reinforce the earlier conclusion [12] that complete auxotrophs can be isolated from Datura innoxia cultures without the use of enrichment-selection methods. The failure to detect revertants of either Ad 1 or Pn 1 after more than a year in culture may be explained in several ways: both auxotrophs could be very stable; any spontaneous revertants produced in culture may not have survived under the cultural conditions used; rare prototrophic revertants did not survive in the presence of large populations of dying auxotrophic cells during revertant-isolation experiments.

Ad 1 and Pn 1 are clearly functionally auxotrophic plant cell strains although the origin and nature of their auxotrophy has not yet been ascertained. The results of growth experiments with some pantothenate and purine nucleotide precursors may indicate that auxotrophy is the result of blocks in the pathways leading to these essential metabolites. Pn 1 grew in the presence of pantoic acid alone but not with fi- alanine or e-ketoisovalerate alone. If the pathways leading to pantothenate formation are the same in Datura cells as they are in microorganisms [13], these results may indicate a block in the pathway leading to pantoic acid formation from c~-ketoisovalerate. The growth of Adl in the presence of AICAR, inosine and, although only slowly, inosinic acid in addition to adenine may indicate a block in the de-novo pathway of purine biosynthesis leading to AICAR formation [9].

In both Adl and Pnl, the observed functional auxotrophy could have an epigenetic origin. Land and Norton [8] reported that the Xan-b 61 mutant of barley, previously thought to be a leucine auxotroph, grew well in the absence of leucine under the right growth conditions. Conditions may be found in which Ad 1 and Pn 1 will grow without the apparent required metabolite. Gavazzi et al. [5] found that their

proline-requiring Zea mays auxotroph grew better in a medium with added proline, arginine and glutamic acid together than with proline alone. They speculated that the mutant could use exogenous arginine to syn- thesize proline, thus bypassing the block in the major biosynthetic route [5]. However, the mutant did not grow in the presence of arginine alone. Such results raise the possibility that cells could contain pools of certain required metabolites at concentrations be- low the threshold value for growth The results of Gavazzi et al. [5] and those of Land and Norton [8] illustrate the fact that only through the use of precise assays to locate enzymatic lesions can the basis of the observed auxotrophy be determined. Such detailed studies for Ad 1 and Pn 1 are underway.

Because both Adl and Pnl are phenotypically auxotrophic, both could be useful in model systems designed to develop new and better methods to obtain auxotrophs. Necessary prerequisites to their use in reconstruction experiments to develop enrichment selection methods for auxotroph isolation would be to determine (a) for how long Ad 1 and Pn 1 can be starved of their required metabolites and still be rescued on supplemented media, and (b) whether cross- feeding would occur between the auxotrophic and wild-type cells when mixed. Enrichment-selection de- pends upon the principle that in a minimal medium auxotrophic cells cease growing whereas wild-type cells continue to grow and can be selectively killed by an agent which affects growing cells only. The rapid development of mammalian somatic cell genetics was in part a results of the ease with which mutant lines were selected in culture following the development of enrichment-selection techniques [11]. The use of similar methods with plant cells has, so far, not led to the isolation of complete auxotrophs [1, 10] although Carlson [2] reported recovering six leaky auxotrophs from cultures of Nicotiana tabacum by applying the technique of 5-bromodeoxyuridine sui- cide-enrichment to mutagenized protoplasts. At- tempts to develop enrichment-selection methods were hampered by the lack of auxotrophic cell lines with which reconstruction experiments could be carried out to test the efficacy of selection techniques.

Pn 1 can be starved of pantothenate and Ad 1 of adenine for 4d and 10d, respectively, and still be rescued when returned to supplemented media. In one experiment. Ad 1 cells were rescued even after 32d of starvation. The results obtained with the double- filter-paper technique of Horsch and Jones [7] indicate that cross-feeding of required nutrients does not occur between wild-type Datura cells and either Adl or Pn 1. The absence of cross-feeding was not a result of the fact that wild-type and auxotrophic cells were separated by a double layer of filter paper since when

484 J. King et al. : Auxotrophic Cell Strains of Datura

the a p p r o p r i a t e r e q u i r e d n u t r i e n t was i n c l u d e d in the

m e d i u m on w h i c h w i l d - t y p e f e e d e r cells w e r e sp read ,

the a u x o t r o p h i c cells a b o v e grew.

T h e resul t s o f o u r s t a r v a t i o n and c ro s s - f eed ing

e x p e r i m e n t s i nd i ca t e t h a t b o t h A d 1 a n d P n l cells

m i g h t be use fu l in a t t e m p t s a t r e c o n s t r u c t i o n .

This work was supported by a grant from the Natural Sciences and Engineering Research Council of Canada to the senior author.

References

1. Aviv, D., Galun, E. (1970) An attempt at isolation of nutritional mutants from cultured tobacco protoplasts. Plant Sci. Lett 8, 299-304

2. Carlson, P.S. (1970) Induction and isolation of auxotrophic mutants in somatic cell cultures of Nicotiana tabacum. Science 168, 487-489

3. Furner, I.J., King, J., Gamborg, O.L. (1978) Plant regeneration from protoplasts isolated from a predominantly haploid suspension culture of Datura innoxia (Mill.). Plant Sci. Lett 11, 16% 176

4. Gamborg, O.L. (1975) Callus and cell culture. In: Plant tissue culture methods, pp. 1-10, Gamborg, O.L., Wetter, L.R., eds. Publ. No. 14383. National Research Council of Canada, Ot- tawa

5. Gavazzi, G., Nava-Racchi, M., Tonelli, C. (1975) A mutation causing proline requirement in Zea mays. Theor. Appl. Genet. 46, 339-345

6. Horsch, R.B., Jones, G.E. (1980) A double filter paper technique for plating cultured plants cells. In Vitro 16, 103-108

7. Horsch, R.B., King, J., Jones, G.E. (1980) Measurement of cultured plant cell growth on filter paper discs. Can. J. Bot., in press

8. Land, J.B., Norton, G. (1970) The nature of the leucine requirement of the barley mutant Xan-b 61. Genet. Res. Camb. 15, 135-137

9. Lehninger, A.L. (1975) Biochemistry, 2nd edn. Worth, New York

10. Polacco, J.C. (1979) Arsenate as a potential negative selection agent for deficiency variants in cultured plant cells. Planta 146, 155-160

11. Puck, T.T., Kao, F-T. (1967) Genetics of somatic mammalian cells. V. Treatment with 5-bromodeoxyuridine and visible light for isolation of nutritionally deficient mutants. Proc. Natl. Acad. Sci. USA 58, 1227-1233

12. Savage, A.D., King, J., Gamborg, O.L. (1979) Recovery of a pantothenate auxotroph from a cell suspension culture of Datura innoxia Mill. Plant Sci. Lett. 16, 367-376

13. Winestock, C.H., Plaut, G.W.E. (1965) The biosynthesis of coenzymes. In: Plant Biochemistry,2nd edn., pp. 391-437, Bon- her, J., Varner, J.E., eds. Academic Press, New York

Received 18 April, accepted l l July I980

Documents

Partial characterization of two stable auxotrophic cell strains of Datura innoxia Mill