Plant Cell Reports
Plant Cell Reports (1989) 8:214-216
© Springer-Verlag 1989
Toxicity of antibiotics on zygotic embryos of white spruce (Picea glauca) cultured in vitro * E. W. T. Tsang 1, H. David 2, A. David 2, and D. I. Dunstan 1 National Research Council of Canada, Plant Biotechnology Institute, 110 Gymnasium Road, Saskatoon, Saskatchewan, Canada S7N OW9, Canada 2 Universit6 de Bordeaux I, Laboratoire de Biologic et Physiologic Vtgttales, Avenues des facultts, F-33405 Talence, France Received November 22, 1988/Revised version received May 2, 1989 - Communicated by F. Constabel
ABSTRACT
a n t i b i o t i c s on woody plants (Young et a l . , 1984).
The t o x i c i t y of kanamycin, hygromycin B, g e n e t i c i n , methotrexate and cefotaxime on zygotic embryos of Picea 91auca was studied. Embryos placed on bud induction medium produced approximately 20 adventitious buds per embryo under control conditions. Addition of a n t i b i o t i c s reduced the number of bud-forming embryos. Using the percentage of embryos with buds as an i n d i c a t i o n of a n t i b i o t i c t o x i c i t y , two-day-old explants were found to be more sensitive than nine-day-old. Kanamycin t o x i c i t y was enhanced by cefotaxime and t h i s e f f e c t increased with increases in concentration of e i t h e r a n t i b i o t i c . Although no morphological difference was observed a f t e r ~I days, embryos growing on medium containing 20 ~g ml -~ kanamycin showed a decrease of 73% in dry weight and 23% in protein content per embryo when compared to control embryos. S i m i l a r l y , a decrease of 38% in dry weight and 40% in protein content per embryolwas found in embryos on medium containing 300 ~g ml- cefotaxime.
In t h i s study, we report the effects of several a n t i b i o t i c s on adventitious bud formation of zygotic embryos of white spruce (Picea 91auca (Moench) Voss) in v i t r o .
ABBREVIATIONS BA, 6 benzyl-aminopurine; EDTA, ethylenediaminet e t r a a c e t i c acid; PVP i 0 , p o l y v i n y l p y r r o l i d o n e (MW I0,000); Tris-HCl, Tris [hydroxymethyl] amino hydrochloride. (
INTRODUCTION The i n t r o d u c t i o n of foreign DNA into c o n i f e r c e l l s by Agrobacterium-mediated transformation of seedlings has been demonstrated in l o b l o l l y pine (Sederoff et a l . , 1986), and Douglas f i r (Dandekar e__t_t a l . , 1987)] However, to our knowledge Agrobacteriumme---diated transformation of regenerable c o n i f e r organs, tissues or c e l l s has not been demonstrated in a manner s i m i l a r to reports f o r angiospermous woody species, such as poplar (Parsons et a l . , 1986) and walnut (McGranahan et a l . , 1988). This is p a r t l y due to the low frequency of regeneration and to the low frequency of ABrobacterium-mediated transformation. For these reasons, a r e l i a b l e method f o r selection that w i l l permit the recovery of transformants at a s u f f i c i e n t l y high frequency is necessary. This requires as a f i r s t - s t e p a knowledge of the r e l a t i v e tolerance of coniferous c u l t u r e material to a n t i b i o t i c s f o r which resistance markers e x i s t . However, l i t t l e is known about the effects of such * N R C C No. 30262 Offprint requests to. E. W. T. Tsang
MATERIALS AND METHODS Preparation of s t r a t i f i e d seeds. S t r a t i f i e d seeds were prepared as reported previously by Toivonen and Kartha (1988). Growth condition of embryos. Excised embryos were cultured, i0 per plate, in d u p l i c a t e , on GMD medium (Mohammed et a l . , 1986) containing 3% sucrose, 0.6% agar and 2.2 ~m BA (pH 5.~) qt 22-24°C, 16h photoperiod (95 ~moles m- s -~) and 30% r e l a t i v e humidity. This medium is referred to as the induction medium. Embryos were allowed to grow on the induction medium f o r a t o t a l of 23 days f o r adventitious bud formation, and then transferred to 3/4 strength hormone-free, GMD medium, containing 0.6% agar, pH 5.6, f o r 21 days to encourage bud elongation. The 3/4 strength, hormone-free, GMD medium is referred to as elongation medium. Bud formation was assessed 44-48 days a f t e r dissection. T o x i c i t y of a n t i b i o t i c s . Excised embryos were incubated on induction medium f o r 2 days to ensure growth, referred to as 2-day-old embryos. A f t e r two days embryos were transferred to induction medium, containing one or more a n t i b i o t i c s , f o r 21 days. They were then transferred to a n t i b i o t i c - f r e e elongation medium f o r an additional 21 days. To compare the influence of explant age on a n t i b i o t i c s e n s i t i v i t y , embryos were grown on induction medium f o r 9 days, before exposure to a n t i b i o t i c - c o n t a i n i n g induction medium f o r 14 days. These are referred to as 9-day-old embryos. The 9-day-old embryos were transferred to elongation medium containing the respective a n t i b i o t i c f o r 7 days before being rescued to a n t i b i o t i c - f r e e elongation medium f o r 14 days. Accordingly, both the 2- and 9-day-old embryos were incubated f o r 21 days on a n t i b i o t i c - c o n t a i n i n g medium and a t o t a l of 23 days on a n t i b i o t i c - f r e e medium. For the additional study on the e f f e c t of kanamycin and cefotaxime, 2 day-old embryos were used. Preparation of a n t i b i o t i c s . A l l a n t i b i o t i c s were dissolved in deionized d i s t i l l e d water. A n t i b i o t i c
215 solutions were f i l t e r - s t e r i l i z e d with 0.22 ~m f i l t e r s (Millex-Gx, M i l l i p o r e Corporation, Bedford, MA, USA). Appropriate q u a n t i t i e s were added to autoclaved medium cooled to 45°C. The a n t i b i o t i c s used were kanamycin, hygromycin B, g e n e t i c i n , methotrexate and cefotaxime. The f i r s t four a n t i b i o t i c s were purchased from Sigma Chemical Co., St. Louis, Me, U.S.A. Cefotaxime was the generous g i f t of Roussel Canada I n c . , Montreal, P.Q., Canada. Concentrations of a l l a n t i b i o t i c s used are described in Table 1. Determination of dry weight and protein content. Embryos were frozen in l i q u i d nitrogen and then l y o p h i l i z e d , p r i o r to measurement of dry weight. Lyophilized embryos were ground in 50 mM Tris HCl b u f f e r (pH 7.2) containing i0 mM EDTA, 5 mM MgCI~, and 2% (w/v) PVP 10. Soluble proteins were assayed by the method of Bradford (1976). RESULTS AND DISCUSSION Development of embryos of white spruce. Growth of control embryos in induction medium was observed as early as 7 days. From i0 to 23 days, embryos grew r a p i d l y (3-4 times the size of the 2-day-old embryos) with prominent swelling around the hypocotyledonary region. From 23 to 30 days, numerous buds were observed. Under control c o n d i t i o n s , 100% embryos formed buds and each embryo produced 20 buds (on average) in 44-48 days (Table 1). Table i. Toxicity of antibiotics to two-day-old and nine-day-old embryos in v i t r o
Embryos w i t h Buds Antibiotics
Control
(#g/ml -I)
(No antibiotic)
Kanamycin
2.5 5.0 i0 20 50
Two
Nine
day-old
day-old
day-old
95
20
23
90 70 35 15 0
16 I0 7 0 0
17 13 8 2 0
50 50 5
45 25 0 0
0
Geneticin
2.5 5.0 i0
40 25 0
Methotrexate
2.5 5.0 I0
a b C
day-old
90 55 i0 0 0
2.5 5.0 i0 20
I00 200 300 400 500
A v e r a g e n u m b e r of b Buds per Embryo Two Nine
i00
Hygromycin B
Cefotaxime
(%)a
Ac A 0 0
A A A 0
45 25 0
A A 0
A A 0
15 0 0
25 i0 0
A 0 0
A A 0
100 i00 90 85 70
i00 95 90 85 80
26 28 16 15 9
27 25 20 13 ii
V a r i a t i o n s b e t w e e n replicates were no greater t h a n i0%. A = a b e r r a n t buds.
eukaryotes and prokaryotes (Gonzalez et a l . , 1978, Pollack et a l . , 1983). Kanamycin appeared to belthe least tox-Tc~f the aminoglycosides. At 5 ~g ~ l - , 55% of the embryos s t i l l bore buds. At 10 ~g ml- , the proportion of embryos which produced buds decreased to 10%. B~d formation on a l l embryos was i n h i b i t e d at 20 ug ml- . Cefotaxime, a ~ - l a c t a m l a n t i b i o t i c , was found to be to~ic above 300 ~g ml- . However, at 100-200 ~g ml- , i t appeared to have no t o x i c a f f e c t on bud formation (Table I ) . The degree of t o x i c i t y in some cases was also reflected in the average number of buds produced per embryo, e.g. f o r kanamycin and cefotaxime. The average number of buds produced per embryo decreased with increases in concentration of these a n t i b i o t i c s . T o x i c i t y of a n t i b i o t i c s on 9-day-old embryos. The effects of the various a n t i b i o t i c s on 9-day-old embryos shown in Table i were consistent with those of 2-day-old. Methotrexate was found to be the most t o x i c with only 25% of embryos producing buds at a concentration of 2.5 ~g m]- . Adventitious buds emerged under these conditions were aberrant I Bud formation was t o t a l l y i n h i b i t e d at i0 ~g ml- . In comparison with the effects on 2-day-old embryos, methot~exate appeared to be less t o x i c at 2.5 and 5.0 ~g ml- with 25% and 10% of embryos respectively to produce aberrant buds. Geneticin and hygromycin B showed s i m i l a r effects on 9-day-old embryos as they did with those of 2-day-old. However, kanamycin was found to be less t o x i c on 9-day-old than 2-day-old emb[yos. Cefotaxime appearedlnontoxic below 300 ~g ml- . At or above 300 ~g ml- , t o x i c i t y occurred. With tobacco protoplasts (Pollock et a l . , 1983) and wheat c a l l i (Mathias and Boyd, 1986-T,~efotaxime was not found to have a s i m i l a r t o x i c e f f e c t . The difference in the response of explants of 2 age classes suggests that bud i n i t i a t i o n in the 2-day-old embryos was more s e n s i t i v e than bud d i f f e r e n t i a t i o n in the 9-day-old embryos to the a n t i b i o t i c s . Effect of kanamycin and cefotaxime. Because kanamycin and cefotaxime were often used in combination in transformation experiments, t h e i r t o x i c i t y were assessed in greater depth in the f o l l o w i n g i n v e s t i g a t i o n s . The t o x i c effects of various concentrations of kanamycin and cefotaxime are shown in Table 2. The combined e f f e c t of the two a n t i b i o t i c s was seen by the reduction in percentage of bud-forming embryos of the combination, compared to each a n t i b i o t i c alone. For example, 55% ~f embryos produced buds in medium containing 5 ~g ml- of kanamycin (and no cefo~axime) and only 10% with the addition of 500 ug ml- of cefotaxime. The use of a combination of a n t i b i o t i c s might therefore a f f e c t the survival of explants a f t e r c o c u l t i v a t i o n experiments and subsequently the p r o b a b i l i t y of scoring the transformants. Table
T o x i c i t y uf a n t i b i o t i c s on the 2-day-old embryos. A l l a n t i b i o t i c s used in the study were found to a f f e c t bud formation to various degrees (Table 1). The most i n h i b i t o r y a n t i b i o t i c appeared to be methotrexate. Methotrexate, a f o l a t e antagonist, i n h i b i t e d bud production on 85% o f l t h e embryos a~ a concentration as low as 2.5 ~g ml- . At 5 ug ml- , 100% i n h i b i t i o n I was observed. Geneticin and hygromycin B at 5 ~g mlreduced bud formation to only 25% and 20% of controls respectively. Adventitious buds induced under thes~ conditions were aberrant (malformed). At 10 ~g ml- , bud production was i n h i b i t e d f o r each a n t i b i o t i c . These two aminoglycosides have been reported to be potent i n h i b i t o r s of protein synthesis in both
2.
Effect of kanamycin two-day-old embryos
and cefotaxime in vitro
on
Percent of embryos bearinq buds a Conc. of cefotaxime (#g ml -l) 0 i00 200 300 400 500 c o
u,
0
10 a Variations
i00
I00
i00
90
85
75
55
55
50
45
30
l0
l0
i0
5
0
0
0
between
replicates
were
no
greater
than
10%.
Analysis of dry weight, protein content and protein synthesis of a n t i b i o t i c - t r e a t e d embryos. The slow growth of embryos made i t d i f f i c u l t to determine the
216 appropriate incubation time required f o r an a n t i b i o t i c to impose i t s affects. Even a f t e r 21 days, no difference in morphology could be discerned between the a n t i b i o t i c - t r e a t e d embryos and the controls (data not shown). For this reason, dry weight and protein content were measured to assess the effects of a n t i b i o t i c s during the 21 day incubation period. Embryos grown on 20 ~g m1-1 kanamycin-containing medium weighed only ~6% of control embryos, and those grown on 300 ~9 ml - cefotaxime weighed only 57%, a f t e r 7 days incubation (Table 3). Comparatively, the protein content of kanamycin-treated embryos was only 75% and cefotaxime-treated was only 83% of the control (Table 3). This indicated that both a n t i b i o t i c s had exerted t h e i r a f f e c t s , resulting in growth i n h i b i t i o n as early as 7 days. The 2-day-old explants were found to have a high i n i t i a l protein content which correlates with the proteinaceous reserve of the embryos (Durzan and Chalupa, 1968). The decrease in protein content in controls a f t e r 2 days was l i k e l y associated with the mobilization of protein reserves during growth. Table 3. Changes in dry weight and protein content of 2-day-old embryos during incubation on medium containing 20 ~g ml -I kanamycin or 300 ~g m1-1 cefotaxime Treatment
Control
(2 days)
Control
(7 days)
Kanamycin Cefotaxime Control
(7 days) (7 days)
(14 days)
Kanamycin Cefotaxime Control
Dry weigbt(DW) Mean Protein content per embryo a DW per embryo b (mg) per embryo (~g per mg DW) (mg)
(14 days) (14 days)
(21 days)
Kanamycin Cefotaxime
(21 days) (21 days)
0.16 0.18 1.65 1.71 1.21 1.03 1.09 0.83 3.77 4.15 2.10 2.04 3.34 3.58 10.13 10.33 2.92 2.56 6.38 6.30
ab Both sets of experiments
0.17 1.68 1.12 0.96 3.96 2. o7 3.46 10.23 2.74 6.34
were done in duplicate,
102.19 104.63 13.36 14.14 10.62 I0.00 11.67 11.09 9.74 10.42 10.04 9.96 9.84 10.04 11.91 12.19 9.66 8.96 7.14 7.30
Mean Protein content per embYro (~g per mg DW) -103.41 13.75 10.31 11.38 I0.08 i0.00 9.94 12.05 9.31 7.22
each with 20-30 embryos
At 21 days, the kanamycin-treated embryos showed a decrease of 73% in dry weight and 23% in protein content. S i m i l a r l y , the cefotaximetreated embryos showed a decrease of 38% in dry weight and 40% in protein content. In conclusion, the t o x i c i t y of several a n t i b i o t i c s on white spruce embryos has been demonstrated. The results showed that these a n t i b i o t i c s affected bud formation and development. With cefotaxime, itlappeared that concentrations at or below 300 ~g ml T M for 21 days should impart minima I t o x i c i t y to the explants. With kanamycin, 20 ~g ml- for 21 days would appear to be an e f f e c t i v e dose for selective d i f f e r e n t i a t i o n of transformants from nontransformants. The other a n t i b i o t i c s tested were considered not to be convenient as selectable markers because of t h e i r undesirable affects on adventitious buds regeneration at the concentrations used. REFERENCES Bradford, M (1976) Anal. Biochem. 72: 248-254. Dandekar, AM, PK Gupta, DJ Durzan and V Knauf (1987) Bio/Tech. 5: 587-590. Durzan, DJ and V Chalupa (1968) Can. J. Bot. 46: 417-428. Gonzalez, A, A Jimenez, D Vazquez, JE Davis and D Schindler (1978) Biochim. Biophys. Acta 521: 459-469. Mathias, RJ and LA Boyd (1986) Plant Sci. 46: 217-223. McGranahan, GH, CA Leslie, SL Uratsu, LA Martin and AM Dandekar (1988) Bio/tech. 6: 800-804. Mohammed, GH, DI Dunstan and TA Thorpe (1986) New Zeal. j . For. Sci. 16: 297-305. Parsons, TJ, VP Sinkar, RF S t e t t l e r , EW Nester and MP Gordon (1986) Bio/tech. 4: 533-536. Pollack, K, DG Barfield and R Shields (1983) Plant Cell Reports 2: 36-39. Sederoff, R, AM Stomp, WS Chilton and LW Moore (1986) Bio/tech. 4: 647-649. Toivonen, PMA and KK Kartha (1988) Plant Cell Reports 7: 318-321. Young, PM, AS Hutchins and ML Canfield (1984) Plant Sci. Lett. 34~ 203-209.
