PlantCeU Reports

Plant Cell Reports (1993) 13:79-82

9 Springer-Verlag 1993

Autoclaved and filter sterilized liquid media in maize anther culture: significance of activated charcoal B. Biiter 1, S . M . Pescitelli 2, K. Berger 1,3, j . E . Sehmid 1, and P. Stamp 1 1 Institute of Plant Sciences, Swiss Federal Institute of Technology (ETH), Eschikon 33, CH-8315 Lindau, Switzerland 2 DowElanco, Biotechnology and Plant Genetics Dept., P.O. Box 68955, Indianapolis, IN, USA 3 P r e s e n t address: Institute of Pharmacy, University of Basel, Kleinhfininger Anlage 3, CH-4057 Basel, Switzerland Received 5 May 1993/Revised version received 1 September 1993 - Communicated by A.R. Gould

Summary. Medium sterilization techniques (autoclaving, filter sterilization and separate sterilization of medium components), combined with preculture exposure to activated charcoal (AC) were evaluated for effects on maize anther culture response. The addition of AC to filter sterilized medium had no effect on the number of embryo-like-structures (ES) produced. For autoclaved medium, pre-culture AC treatment resulted in a 3-fold increase in ES yield over medium lacking AC. When AC was included, autoclaved medium was more productive than filter sterilized medium. Autoclaved media without AC gave lower response than filter sterilized medium. Separate sterilization of sucrose or FeEDTA was beneficial for media autoclaved in the absence of AC. However, when all components were autoclaved together in the presence of AC, there was no advantage to separate sterilization. The maximum ES frequency (224.6 ES/100 anthers) was obtained with the genotype ETH-M 52 cultured in autoclaved medium which had been exposed to AC (5 g/L) for 96 h prior to culture initiation. It is supposed that the higher ES frequencies observed with AC-treated, autoclaved media were due to a) the availability of glucose and fructose following heat-induced hydrolysis of sucrose and b) the AC-mediated adsorption of inhibitory compounds produced during autoclaving.

Key words: Maize - Anther culture - Autoclaving Medium toxicity - Activated charcoal Introduction: Activated charcoal (=AC) is an important component of anther culture induction media (KOHLENBACH and WERNICKE, 1978; GENOVESI and COLLINS, 1982; JOHANSSON, 1983). One of the primary benefits appears to be the adsorption of inhibitory substances in the culture medium. Toxic compounds originate C o r r e s p o n d e n c e to:

B. Biiter

from certain media components, particularly sucrose, which breaks down when subjected to high temperature during autoclave sterilization. One such compound, 5(hydroxymethyl-2-furaldehyde) has been identified as a breakdown product of monomeric saccharides (THEANDER and NELSON, 1988) and causes severe reduction in anther culture efficiency (WEATHERHEAD et al., 1979). Other inhibitory substances such as ethylene (HORNER et al., 1977) and phenolic compounds (WEATHERHEAD et al., 1979) are produced by the anther tissue during culture and may also be adsorbed by AC, thereby improving growth conditions. In addition to the beneficial effects, the use of AC in culture media presents complicating factors. Since the adsorptive function is non-specific, it is uncertain which components in the medium are actually available (GENEVESI, 1990). Beneficial us well as toxic compounds may be adsorbed (FRIDBORG et al., 1978; EBERT und TAYLOR, 1990). Moreover, the presence of AC in liquid media obscures visual observation, and AC adheres to tissue surfaces which may result in a decrease in haploid induction (WERNICKE and KOHLENBACH, 1976; TYAGI et al., 1980). Filter sterilization (CHU and HILL, 1988) and separate autoclaving of media components (SCHENK et al., 1991) has been used for liquid culture media in order to avoid or minimize the presence of heat-induced toxic compounds thereby reducing the need for AC. PESC1TELLI et al. (1989) removed the AC after autoclaving, prior to culture initiation, however, did not report on the effects of alternative sterilization methods. In the present study we have investigated the use of different medium sterilization techniques (autoclaving, filter sterilization and separate sterilization of medium components) in combination with exposure to AC during the period prior to culture initiation and evaluated the effects on the production of embryo-like-structures (=ES) in maize anther culture.

80 Materials and Methods: Two genotypes were used in the present study: PA91xFR16 (Dr. Joe Petolino, Dow Elanco, Indianapolis, USA) and ETH-M 52 ('Dfis hybrid was produced on the experimental station of ETH in Esdfikon, Switzerland, by intermating an $1 of the single cross S H A O - P A TANGxSHUI-BE with the inbred PA-TANG-BE; the single cross and the inbred were provided by Dr. Li Dawei, Institute of Genetics, Beijing, Chins.). All donorplants were grown in the greenhouse during the period from March to June 1991 (25/20~ • 3~ 16 h light). Tassel harvest, cold pretreatmenL stage determination, inoculation and incubation followed previously published procedures (BLYI'F.Ret al., 1991). Immediately after inoculation, all cultures were subjected to a 14~ post-plating treatment for 4 days. The induction medium consisted of YP major salts (KU et al., 1978) with the addition of 0.1 mg/L triiodobenzoic acid, 0.25 mg/L L-thiamine-HC1, 1.3 mg/L nicotinic acid, 7.7 mg/L glycine, 125 mg/L L-proline, 500 mg/L casein acid hydrolysate and 90 g/L sucrose. All media were sterilized either by autoclaving (1.2 bar, 20 rain.) or filter sterilizing (Milli!mqe Sterivex GS filter, 0.22 pm, 1.0 bar). The presence of AC was limited to the period of medium preparation, prior to the initiation of culture. AC was removed from all autoclaved media 24 h after autoclaving, unless otherwise stated. Autoclaved media not containing AC were also filter ste'rdized. In the first experiment (Exp. A), sterilization methods (autoclaving vs. filter steriliz/ng) were compared for media containing AC (5 g/L) during the preculmre period and media without AC (Tab. 1). Duration of exposure to AC was also investigated. The effects of different AC concentrations in the range from 0 to 10 g/L were evaluated in a second experiment (Exp. B, Fig. 2). A third experiment (Exp. C) was performed to analyze the effects of separate sterilization of the medium components FeEDTA (=FeS04 + Titriplex I l l Merck) and sucrose, respectively. In this experiment (Fig. 3), Treatments I-IV did not contain AC and included: I) separate sterilization of FeEDTA by autoclaving; H) separate sterilization of sucrose by autoclaving; m ) separate sterilization of sucrose by filter sterilizing; IV) autoclave sterilization of all components together. In Treatment V, the medimn contained 5 g/L AC and all components were autoclaved together (AC was removed 24 h after autoclaving). Inall treatments, the main portion of the medium was sterilized by autoclaving, regardless of the sterilization method of individual components. In all experiments, ES larger than 0.5ram (Fig 1 a) were counted 4, 6 and 8 weeks after culture initiation and then transferred to either regeneration medium (=RM)(BOTER et aL, 1991) or a modified N6 callus induction medium (=CM) (LUPPOTID and LUSARDI, 1988) containing 2.5 mg/L 2,4-D, 25 g/L sucrose and 2.7 g/L phytagel instead of agar. ES on CM were kept in the dark for 4 weeks and then evaluated for the formation of regenerable callus (=RC). RC was defined as callus displaying a eOml~Ct nodular appearence (Fig. 1 b). RC transferred to RM were evaluated for shoot and root development (Fig 1.c). Phntlets 2-3 cm in size (Fig. 1 d) were c.~mted and transferred to a roofing medium consisting of YP major salts (KU et al., 1978), 2.5 mg/L NAA, 25 g/L sucrose and 6.5 g/L agarose.

Results: In EXP. A, the addition of AC to the filter sterilized medium had no effect on the number of ES produced for the genotype PA91xFR16 (Treatment I vs. II, Tab. 1). In contrast, the presence of AC in autoclaved medium had a significant effect on ES yield. Anthers cultured in medium exposed to AC during autoclaving,

Fig. 1: Production of doubled haploid seeds via regenerable callus: a. anthers with a high ES production; b. callus with regenerable structures = regenerable callus (= RC); c. shoot proliferation on RC; d. growth and root formation on rooting medium; e. regenerated donbled haploid plant after selling; f. doubled haploid seeds.

Table 1 (Experiment A): Comparison of autoclaved (AU) and illtersterilized (FS) induction media in maize anther culture: effects of activated charcoal (AC)[*I] (ES = embryo-like structures; NT = Not tested; RC = regenerable calli; SE = standard error;, 390 anthers/treatment). rreat~ent

AU or FS

+/AC

Time ES/100anth. Plants/ RC/ AC (+ SE) 100 ES (:I:SE) expe . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . sure PA91x ETH PA91x ETH [h] FR16 M 5 2 FR16 M 5 2

I

FS

-AC

---

53.1 (9.3)

NT

0.5 (0.5)

NT

II

FS

+AC

24

52.6 (17.2)

NT

-----

NT

]II

AU

-AC

---

42.7 (11.3)

NT

0.7 (0.7)

NT

IV

AU

+AC

0[*2]

67.7

NT

2.7

NT

(14.1)

(1.2)

V

AU

+AC

24

111.5 133.9 2.3 (24.6) (23.4) (1.0)

20.0 (2.3)

VI

AU

+AC

96

NT

20.4 (2.3)

224.6 NT (30.7)

*1: In all treatments: AC removed prior to anther inoculation. *2: AC was removed immediately after autoclaving.

81 200 [~

E8/100 anthers

I

RC/IO0 E8

T

150 I

...... ..,....~.=

iiiiii! ii!iii!! i!ii!ii!l

1 O0

5o

:i~::ii~::

0

............

1

0.1

2.5

5

iii!ilililil

10

Activated Charcoal (g/L)

Fig. 2: Impact of a 24 h presence of activated charcoal (= AC) subsequent to autoclaving of the induction medium on the frequency of embryo-like-structures (= ES) and regenerable ealli (= RC): comparison of different AC concentrations (Genotype: ETH-M 52; 300 anthers/treatment; error bars = standard error).

ES/IO0 anthers 300 260

I

R C / I O 0 ES

FsEDTA sutoelsved sspsratsty

Sucrose autoclaved separately

Sucrose filter sterll. separately

All compoIteflts eutoolaved

All r ~ Rents auto-

-AC

-AC

-AC

-AG

*AO

olawd

200 150 100 50 0

I

~v II

dium lacking AC (Tab. 1), however, ES yield was higher in medium exposed to AC for 24 h after autoclaving. For the genotype ETH-M 52, the effect of AC appeared to be enhanced when post-autoclave exposure to AC was increased from 24 to 96 h (Tab. 1). In medium lacking AC, ES frequency in filter sterilized medium was higher than in autoclaved medium; when AC was included, autoclaved medium was more productive

(Tab. 1). The effect of AC concentration on ES production in the genotype ETH-M 52 was evaluated for medium exposed to AC during autoclaving with subsequent removal after 24 h (Exp. B). The number of ES/100 anthers increased at higher AC concentrations (Fig. 2). The maximum yield was observed at 10 g/L, a 3-fold increase over the control treatment (0 g/L). In the absence of AC, separate autoclave sterilization of either sucrose or FeNaEDTA resulted in higher ES production than medium in which all components were autoclaved together (Exp. (2) (Fig. 3, Treatment I and II vs. IV). Separate sterilization of sucrose by filtering was less effective than separate sterilization by autoclaving (Fig. 3, Treatment I vs. III). The highest ES production was observed in the medium in which all components were autoclaved together in the presence of AC with removal after 24 h (Fig. 3). In all experiments, the regeneration of plants from ES appeared to be uneffccted by sterilization method or AC treatment. Overall, formation of RS from ES was low for the genotype PA91xFR16. The frequency of RC formation for ETH-M 52 was approximately 20% in most treatments. Several hundred plants were transferred to the greenhouse (Fig. 1 e) where 10-20% produced doubled haploid seed (Fig. 1 f) (data not shown).

Discussion

III

IV

v

Treatment

Fig. 3.: Separate sterilization of FeEDTA and sucrose in autoclaved induction media without activated charcoal (= AC): effects on the formation of embryo-like-structures (= ES) and regenerable ealli (= RC) (AU = autoclaved; FS = ~ t e r sterilized; Genotype: ETH-M 52; 300 anthers/treaunent; error bars = s ~ error).

with subsequent removal after 24 h, produced 3x more ES/100 anthers than medium lacking AC (Tab. 1, Treatment V vs. III). The duration of exposure to AC after autoclaving was also important for ES production. Exposure to AC during autoclaving, followed by immediate removal, improved response compared to me-

AC is required for maximum response in maize anthers cultured on semi-solid medium (GENOVESI and COLLINS, 1982). However, the presence of AC in the liquid medium can have a negative effect on embryo growth (WERNICKE and KOHLENBACH, 1976; TYAGI et al., 1980) and complicates visual observation of the culture. Filter sterilization may obviate the need for AC in liquid medium by avoiding the high temperature induced breakdown of media components during autoclaving. In the absence of AC, CHU and HILL (1988) found higher pollen embryo yield in filter sterilized vs. autoclaved liquid media. However, the resuits of the present study indicate that a higher response is obtained from autoclaved vs. filter sterilized medium when AC is included during the preeulture period. In contrast to media sterilized by filtering, the sucrose in autoclaved media is partially hydrolized to glucose and fructose (BALL, 1953; SINGHA et al.

82 1987); the extent of this heat dependent hydrolysis is further increased in the presence of AC (DRUART and DE WULF, 1993). No biochemical analysis was performed, but it seems possible that the availability of glucose and fructose contributed to the better performance of the autoclaved medium. In addition to the heat-induced hydrolytic breakdown to glucose and fructose, exposure of sucrose to high temperature conditions during autoclaving also produces toxic substances (WEATHERHEAD et al., 1978; THEANDER and NELSON, 1988). Maize anther culture media typically contain high concentrations of sucrose, therefore, these by-products may reach inhibitory levels after autoclave sterilization. Results from the current study indicated that pre-culture exposure of the medium to AC improved ES production significantly, suggesting that these deleterious compounds may have been removed. The beneficial effect of AC was observed with a relatively short exposure time (24-96 h after autoclaving). Removal of the AC prior to culture did not appear to effect the production or development of ES. It was previously shown (Bt2TER, 1992) that the presence of AC during the culture period did not essentially improve response. It has been suggested that AC may also function to remove deleterious by-products resulting from the breakdown of the anther wall during culture (GENEVESI, 1990), however -at least in liquid media- this appears to be a minor effect. The concentration of AC commonly used in semisolid medium for maize anther culture is 5 g/L (GENOVESI, 1990). For liquid medium in which the AC is removed prior to culture, we found that ES-production increased with increased concentrations of AC up to 10 g/L. Levels of AC above 10 g/L were cumbersome to use and difficult to remove by filtration. As suggested in Exp. A, extending the time of exposure to AC led to improved ES production. Prolonged exposure prior to culture may be an effective alternative to the use of high concentrations of AC, particularly in routine culture. In a recent report, SCHENK et al. (1991) demonstrated that the breakdown of sucrose during autoclaving can be catalyzed by other components present in the medium, especially FeEDTA. Their results indicated that autoclaving either sucrose or FeEDTA separately from the other medium components, reduced the heat-induced toxicity of the culture medium. In the present study, we found that -in the absence of AC- autoclaving of FeEDTA or sucrose separately yielded higher numbers of ES than a medium in which all components were autoclaved together. These results concur with SCHENK et al. (1991) and suggest that higher concentrations of toxic compounds were present in Treatment I (Fig. 3) due to the FeEDTA-catalyzed breakdown of sucrose. However, the presence of AC during autoclaving appears to ameliorate these effects

and precludes the need for separate sterilization of medium components. In conclusion, the addition of AC to autoclaved liquid medium and subsequent removal prior to the culture period, provided for maximum ES production in maize anther culture compared to other methods of sterilization. These results may be relevant to other areas of plant tissue culture where high sucrose levels are required.

This publication is dedicated to Prof. era. Dr. Dr.h.c.F.W. Schnell's 80 th birthday. Acknowledgements. We wish to acknowledgeK. Sigrist-Htneisen and E. Ganmerfor technical assistance.

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