Plant Cell Reports

Plant Cell Reports (1987) 6:454-457

© Springer-Verlag 1987

The effect of cefotaxime on the growth and regeneration of callus from four varieties of barley (Hordeum vulgate L.) R. J. Mathias and C. Mukasa Cytogenetics Department, Plant Breeding Institute, Institute of Plant Science Research, Marls Lane, Trumpington, Cambridge CB2 2LQ, UK Received September 2, 1987 / Revised version received October 26, 1987 - Communicated by H. L6rz

ABSTRACT Recently it has been reported that the cephalosporin antibiotic cefotaxime increases growth, regeneration and embryogenesis in wheat calli. We investigated the effect of cefotaxime on callus initiated from immature embryos of four barley (Hordeum vulgare L.) varieties. In calli cultured in the presence of antibiotic callus growth was up to 45% greater than in controls and the frequency of regenerating calli was increased by up to 80%. There was an apparent interaction of the antibiotic with genotype and the 2,4-D in the medium.

INTRODUCTION A number of antibiotics and clinical and agriculture fungicides have been tested in tissue cultures as a means of controlling or eliminating contaminating micro-organisms (De Ropp 1949, Umiel and Goldner 1976, Dodds and Roberts 1981, Phillips et al. 1981, Brown et al. 1982, Pollock et al. 1983, Shields et al. 1984, Hauptmann et al. 1985). The most common experience has been that these compounds are toxic to plant cell cultures, but in a few cases some improvement in growth has been reported (De Ropp 1949, Phillips et al. 1981, Pollock et al. 1983). The use of antibiotics in culture has recently become more widespread with the emergence of i) antibiotic resistance genes as selectable markers in transformation experiments (L6"rz et al. 1985, Pietrzak et al. 1986, Hille et al. 1986, Lloyd et al. 1986) and li) transformation systems based on the co-cultivation of Agrobacterium tumefaciens with explants and cultured cells (Fraley et al. 1984, McCormick et al. 1986, Parsons et al. 1986). The aminoglycoside kanamycin is commonly used to select for plant cells transformed wit ~ the neomycin phosphotransferase marker gene (L~rz et al. 1985, Hain et al. 1985, Pietrzak et al. 1986). The antibiotic is inhibitory to cell growth at comparatively low concentrations (50-100 ~g/ml), however Owens (1979) has reported that at very low concentrations (2-8 ~g/ml) kanamycin promotes the regeneration of tobacco and carrot cultures. In testing cefotaxime as a means of eliminating A. tumefaciens from wheat calli in co-cultivation experiments Mathias and Boyd (1986) observed that in the presence of low levels of antibiotic (60-100 ~g/ml) there was a dramatic improvement in the callus culture response.

Offprint requests to: R. J. Mathias

In vitro culture of the small grain cereals remains a difficult area (for reviews see Schaeffer et al. 1984, Koblitz 1986). In order to examine whether the effect of cefotaxime was specific to wheat or might have applications in improving the response of other cereals we tested its effect on four varieties of barley. There was a significant improvement in the growth and regeneration of calli in the presence of cefotaxime and an apparent interaction of the antibiotic with the genotype and the medium. MATERIALS AND METHODS The plant varieties Apex, Blenheim, Everest and Porter used in these experiments were inbred lines of Barley (Hordeum vulgare L.) maintained at the Plant Breeding Institute, Cambridge, U.K. Grains were imbibed on water soaked filter papers in Petri dishes at 4°C for 3 days and then germinated at 2 0 % in the dark. Four day-old seedlings were potted into John Innes No. 1 compost in 'Jiffypots' and vernalised for 4 weeks at 4°C. Plants were then potted into 4-inch pots and grown to maturity in a greenhouse at 20°C ± 3°C, under 16-h daylength. In these varieties anthesis occurred while most of the spike was still enclosed in the 'boot'. As spikes reached maturity they were checked daily and bagged and dated on the day of anthesis. The spikes were harvested when the scutellum of the oldest embryos in the ear was 1 mm long. The grains from the florets around the middle of the ear were surface sterilised for 15 minutes in 10% Domestos (Lever Bros., U.K.) and washed in two changes of sterile distilled water. The excised embryos were plated on initiation medium with the embryo embedded in the medium. The basal medium was modified from Murashlge and Skoog (1962) as detailed by Sears and Deckard (1982). The initiation, maintenance and regeneration media contained the basal medium with 1.0 mg/l, 0.5 mg/l and 0. i mg/l of 2,4-D respectively. Cefotaxamine (Calbiochem) dissolved in water, was filter sterilised and added to the autoclaved medium immediately before the plates were poured. In the autoclaved antibiotic treatments the antibiotic was added to the medium before it was autoclaved. The cultures were maintained under continuous illumination at 27°C ± I°C. The calli were subcultured

at 2 week intervals.

After

455 Table i. Analysis of variance for callus fresh weights, shoot primordia formation and shoot regeneration of four barley varieties on initiation, maintenance and regeneration medium (significance levels *:5%, **:1%, ***:0.01%)

df

Apex ms

Blenheim ms

treatment residual

3 32

0.007904 0.0004781

0.00021157* 0.00005731

0.0004182* 0.0001232

0.0002825 0.0006205

Maintenance medium

treatment residual

3 32

0.095383*** 0.005138

0.042700** 0.007725

0.040837** 0.006144

0.064236*** 0.005593

Regeneration medium

treatment residual

3 32

0.102036*** 0.005561

0.021309* 0.007025

0.047192** 0.004974

0.076726*** 0.005490

treatment residual

3 39

0.52714*** 0.02271

0.12737*** 0.01494

0.12537* 0.03971

0.21097*** 0.01771

Maintenance medium

treatment residual

3 39

0.053984*** 0.02298

0.24284*** 0.02265

0.22189*** 0.01364

0.28013*** 0.02303

Regeneration medium

treatment residual

3 33

0.63220*** 0.02405

0.32476*** 0.01763

0.434551*** 0.007955

0.40333*** 0.01542

Initiation medium

treatment residual

3 39

0.09498* 0.01954

0.020037 0.007486

0.01959 0.01190

0.05993** 0.01003

Maintenance medium

treatment residual

3 39

0.33268*** 0.01970

0.09323* 0.01750

0.37410*** 0.01671

0.22162*** 0.01489

Regeneration medium

treatment residual

3 39

0.48152*** 0.02026

0.38019*** 0.015945

0.58179*** 0.01102

0.45857*** 0.01920

Callus fresh weight Initiation medium

Shoot p r i m o r d i a Initiation medium

Everest ms

Porter ms

Shoot r e g e n e r a t i o n

4 weeks on initiation medium they were transferred to maintenance medium and 4 weeks later to regeneration medium. After 4 weeks on each medium the average fresh weight of callus was recorded and the number of calli with shoot primordia (green spots) and leafy shoots scored. Calli were also examined under the microscope for embryoid formation. The data was analysed by analysis the individual petri dishes as treatment consisted of i00 calli.

of variance replicates,

using each

RESULTS The growth rates and frequencies of shoot primordia and shoot formation were very similar among the untreated control calli of the four barley varieties. Although there was no dramatic intervarietal differences Blenheim was consistently less responsive than the other varieties. The overall analysis of variance for the mean fresh weights of calli after one month on initiation, maintenance and regeneration medium is presented in Table !, the means are depicted in Figure i. Cefotaxime promoted callus growth of all the varieties but the precise pattern of response differed between the varieties. The most significant effects were on maintenance and regeneration medium where the fresh weights of antibiotic treated calli were up to 45% greater than the controls. Callus growth of Apex, Everest and Porter was stimulated by cefotaxime at 60 and i00 ~g/ml but Blenheim responded only on 60 ~g/ml. Autoclaved cefotaxime was eithe~ neutral or inhibitory in its effect, except for one instance where growth of Everest, on initiation medium, was stimulated by autoclaved antibiotic. The analysis of variance of the mean percent of calli forming shoot primordia is presented in Table i. Culturing calli on cefotaxime significantly affected the frequency of shoot primordia formation. Antibiotic at 60 ~g/ml increased the number of calli with primordia in all varieties by up to 75% over the

controls. At I00 gg/ml, morphogenesis in Apex and Blenheim was increased by up to 67%, but this concentration of antibiotic was neutral or inhibitory in Everest and Porter. Autoclaved antibiotic inhibited primordia formation in all varieties and on most media. The mean percent of calli with shoots are depicted in Figure 2 and the analysis of variance for shoot regeneration frequencies on initiation, maintenance and regeneration medium is given in Table I. The frequency of shoot regeneration from all varieties was increased on 60 ~g/ml of cefotaxime, up to 80% over controls in some instances. The effect of i00 ~g/ml of antibiotic was not so consistent as 60 ~g/ml, only in t h e variety Porter were the effects of the two concentrations of antibiotic similar. Autoclaved antibiotic frequently inhibited regeneration, in some cases causing a three-fold reduction in the frequency of shoot formation. Embryogenic regions were observed on calli of all varieties but the occurrence of these regions was sporadic and no effect of antibiotic on embryogenic callus formation was detected. DISCUSSION The results presented in this paper demonstrate that cefotaxime promotes growth and morphogenesis in callus cultures of barley. The addition of antibiotics to cell cultures, particularly at bacteriostatic or bactericidal concentrations, frequently results in inhibition of growth and development (Umiel and Goldner 1976, Dodds and Roberts 1981, Shields et al. 1984). However improved in vitro responses in the presence of antibiotic have also been described (De Ropp 1949, Owens 1979, Pollock et al. 1983, Mathias and Boyd 1986). As antibiotics have commonly been used to try to eliminate bacterial contaminants it may be difficult to identify whether improved responses in culture are due to the 'direct' physiological action of the antibiotic on the cells or an indirect effect on contaminating organisms.

456 BLENHEIM

APEX

0.6

0.6 0.5

0.5

< \ \ \ \ \ \ \ \ \ \

gms 0.4 0.3 0.2 0.1

0

60

gms 0.4

1i 100

0.3 0.2 \ 0.1 \

IOOA

0

ug/ml

EVEREST

PORTER

0.5

0.5

60

100

IOOA

ug/ml

100

IOOA

ug/ml

0.4

0.4 gms

gms 0.3

0.3

0.2

0.2

0.1

0.1

60

100

IOOA

N

60

ug/ml

Figure i. Mean fresh weights of calli of four barley varieties on initiation, medium, with and without cefotaxime

maintenance

and regeneration

Black bars: initiation medium, hatched bars: maintenance medium, open bars: regeneration medium. 100A = i00 gg/ml of autoclaved antibiotic. Starred treatments are significantly different from the control on the same medium (significance levels *:5%, **:1%, ***:0.1%). BLENHEIM

APEX

100

100 %

% 8O

80

60

6O

40

4O

20

20 O

0 0

60

100

100A

0

ug/ml

60

100

IOOA

ug/ml

60

100

IOOA

ug/ml

pORTER

EVEREST

100

100 %

%

80

80 60 40

:

20

2o

0 0

Figure 2. cefotaxime

Percent of calli producing

60

100

IOOA

ug/ml

0

shoots on initiation, maintenance

and regeneration medium, with and without

Black bars: initiation medium, hatched bars: maintenance medium, open bars: regeneration medium, lOOA = i00 ~g/ml of autoclaved antibiotic. Starred treatments are significantly different from the control on the same medium (significance levels *:5%, **:1%, ***:0.1%).

457 The aminoglycosides kanamycin and streptomycin improve regeneration of shoots from tobacco and carrot (Owens 1979), and beta-lactams - both penicillins and cephalosporins - are reported to 'potentiate' the growth of protoplast derived cell colonies of Tobacco (Pollock et al. 1982). Both are apparently 'direct' effects on cell metabolism. Cefotaxime, and to a lesser extent carbenicillin, have significant and apparently 'direct' effects on the in vitro response of wheat callus (Mathias and Boyd 1986). This report describes similar effects of cefotaxime on the growth and regeneration of four varieties of barley. It is unlikely that the effect is due to antibiotic action on endogenous bacterial infections. We have never observed systemic infections in any wheat, maize or barley cultures initiated from immature embryos or succeeded in growing bacteria from apparently axenic barley cultures incubated in bacterial culture medium. The antibacterial action of the beta-lactams results from their affinity for the penicillin binding proteins (PBPs) on the outer surface of the bacterial envelope. The PBPs are peptidoglycan transpeptidases involved in the final stages of bacterial cell wall synthesis (Selwyn 1980). Cefotaxime specifically and irreversibly binds PBP-3, a transpeptidase essential for normal cell division in E. coli (Curtis et al. 1979). However the transpeptidation step is unique to bacterial cell wall synthesis (Selwyn 1980) so it is unlikely that the effect of the antibiotic in plant cells is related to its antibacterial mode of action. While the effect of cefotaxime on plant cells is unlikely to result from PBP binding the fact that both penicillins and cephalopsorins have been reported to affect the growth and morphogenesis of cultures suggests some specificity of action. Some cephalosporins must act on alternative, or additional, target sites to the PBPs as certain cephalosporins are active against mycoplasmas which do not have cell walls (O'Callaghan and Muggleton 1972). An obvious explanation for the activity of cefotaxime in culture is that the molecule mimics a plant growth regulator. Shoot regeneration from wheat callus cultures has been increased by the addition of gibberellic acid (Mathias and Atkinson 1987) and cytokinin (Ahloowalia 1982, Papenfuss and Carman 1987) to media containing 2,4-D. However in a conventional bioassay which is able to detect, and discriminate between, auxin, cytokinin and gibberellin activity (Wright 1968) cefotaxime did not mimic the activity of any of these growth regulators (authors unpublished data). The possibility that cell metabolism converts cefotaxime to a compound with growth regulator activity was discussed by Mathias and Boyd (1986). Mathias and Boyd (1986) described the toxic effects of autoclaved cefotaxime on wheat calli. We observed a similar effect in barley, except in one instance where autoclaved cefotaxime actually promoted callus growth of Everest on initiation medium. The thermal decomposition products of cefotaxime and thus the chemical basis of these toxic effects are not known. Cefotaxime has a significant 'direct' effect on the culture response of barley callus. There is an interaction of genotype with the antibiotic since the varieties differ in their response to cefotaxine. There also seems to be an interaction of 2,4-D with antibiotic as the effect of cefotaxime depends on the

background medium and these differ only in 2,4-D content. Although the mechanism of the cefotaxime effect on wheat and barley callus is unknown it may be of. value in promoting regeneration in these species, particularly among recalcitrant genotypes.

REFERENCES Ahloowalia BS (1982) Crop Sci. 22:405-410 Brown DM, Groom CL, Cvitanik M, Cooper JL, Arditti J (1982) Plant Cell Tissue Organ Cult. 1:165-180 Curtis NA, Orr, D, Ross GW, Boulton MG (1979) Antimicrob. agents and Chemotherap. 16:533-539 Fraley RT, Horsch RB, Matzke A, Chilton MD, Cilton WS, Sanders PR (1984) Plant Mol. Biol. 3:371-378 De Ropp RS (1949) Phytopathology 39:822-828 Dodds JH, Roberts LW (1981) In Vitro 17:467-470 Hain R, Stabel P, Czernilofsky AP, Steinbiss HH, Herrera-Estrella L, Schell J (1985) Mol. Gen. Genet. 199:161-168 Hauptmann R, Widholm JM, Paxton JD (1985) Plant Cell Rep. 4:129-132 Hille J, Verheggen F, Roelvink P, Franssen H, Van Kammen AB, Zabel P (1986) Plant Mol. Biol. 7:171-176 Koblitz H (1986) In: Bajaj YPS (ed) Biotechnology in Agriculture and Forestry, vol 12, Springer-Verlag Berlin pp 181-203 Lloyd AM, Barnason AR, Rogers SG, Byrne MC, Fraley RT, Horsch RB (1986) Science 234:464-466 Lorz H, Baker B, Schell J (1985) Mol. Gen. Genet. 199:178-182 Mathias RJ, Boyd LA (1986) Plant Sci. 46:217-223 Mathias RJ, Atkinson E (1987) Theor. Appl. Genet. (in press) McCormick S, Niedermeyer J, Fry J, Barnason A, Horsch R, Fraley R (1986) Plant Cell Rep. 5:81-84 Murashige T, Skoog F (1962) Physiol. Plant. 15:473-497 O'Callaghan CH, Muggleton PW (1972) In: Flynn EH (ed) Cephalosporins and Penicillins chemistry and biology, Acad. Press NY, pp 438-495 Owens LD (1979) Plant Sci. lett. 16:225-230 Papenfuss JM, Carman JG (1987) Crop Sci. 27:588-593 Parsons TJ, Sinkar VP, Stettler RF, Nester EW, Gordon MP (1986) Biotechnology 4:533-536 Phillips R, Arnott SM, Kaplan SE (1981) Plant Sci. Lett. 21:235-240 Pietrzak M, Shillito RD, Hohn T, Potrykus I (1986) Nuc. Acids Res. 14:5857-5868 Pollock K, Barfield DG, Shields R (1983) Plant Cell Rep. 2:36-39 Sears RG, Deckard EL (1982) Crop Sci. 22:546-550 Selwyn S (1980) In: Selwyn S (ed) The Beta-lactam antibiotics, Hodder and Stoughton, London pp 56-90 Schaeffer GW, Lazer MD, Baenziger PS (1984) In: Sharp WR, Evans DA, Ammirato PV, Yamada Y (eds) Handbook of Plant Cell Culture vol. 2, Macmillan NY, London pp 108-136 Shields R, Robinson SJ, Anslow PA (1984) Plant Cell Rep. 3:33-36 Umiel N, Goldner R (1976) Protoplasma 89:83-94 Wright STC (1968) In: VI International conference on plant growth substances, Carleton University, Runge Press Ottawa pp 521-542.

ACKNOWLEDGEMENT C .M. was Company.

supported

by

the

Agricultural

Genetics

The effect of cefotaxime on the growth and regeneration of callus from four varieties of barley (Hordeum vulgare L.).

Recently it has been reported that the cephalosporin antibiotic cefotaxime increases growth, regeneration and embryogenesis in wheat calli. We investi...
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