1021-4437/04/5104- © 2004

MAIK “Nauka

/Interperiodica”0559

Russian Journal of Plant Physiology, Vol. 51, No. 4, 2004, pp. 559–562. Translated from Fiziologiya Rastenii, Vol. 51, No. 4, 2004, pp. 621–625.Original Russian Text Copyright © 2004 by Danilova, Dolgikh.

INTRODUCTION

To induce morphogenesis

in vitro

, phytohormonesand hormone-like synthetic growth regulators are com-monly used. However, nontraditional inducers of mor-phogenesis, such as oligosaccharins, some amino acids[1], light fluence rate and quality [2], weak electric cur-rent [3], and others stimuli are also effective for regen-eration of some plant genotypes. Thus, xyloglucan pen-tasaccharide stimulated morphogenesis in culturedwheat tissues [4], whereas trisaccharide favored cottonsomatic embryogenesis [5]. The extract from ginsengrhizoflora (emistime) was very effective in the stimula-tion of shoot regeneration from maize callus [6].

A stimulatory effect of some antibiotics, cefotaximein particular, on growth and morphogenesis of culturedtissues was also shown. Cefotaxime is a semisyntheticanalog of cephalosporin, the third generation antibiotic.Cephalosporins are secreted by fungi from the genus

Cephalosporum.

The main producer of this antibiotic isthe fungus

C. acremonium.

Cephalosporin is a memberof the

β

-lactam group being related to penicillin in itschemical nature [7]. Cephalosporin antibiotics areeffective at relatively low concentrations; they have arather wide spectrum of biological activity and mini-mum toxicity on eukaryotes. Two of these antibiotics,carbenicillin and cefotaxime, are widely used for

Agro-bacterium tumefaciens

elimination in gene engineeringexperiments [8]. The effects of these antibiotics on cal-lus growth and morphogenesis were noted for someplant species. Thus, in apple callus, 200 mg/l cefo-taxime improved regeneration and shoot developmentfrom apical buds; at a concentration of 500 mg/l, itinduced the development of malformed shoots. Carbe-

nicillin (500 mg/l) suppressed shoot formation andenhanced callus growth [9]. In cotton embryo axes,cefotaxime stimulated or inhibited shoot developmentdepending on the cultivar [10]. In tobacco, cefotaximeweakly inhibited shoot formation from cotyledonaryexplants and rooting of regenerants obtained from leafdisks [11]. When cephalosporin affect dicotyledonousplant tissues in a different way, cefotaxime and carbe-nicillin exerted only a stimulatory action on cereals.Mothias and Boyd [12] and Mothias and Mukasa [13]reported a promotion of callus growth and an enhance-ment of organogenesis in cultured barley and wheat tis-sues. Barelli

et al.

[14] observed an increased frequencyof embryogenic callus formation and plant regenerationin durum wheats; in low-morphogenic cultivar, thenumber of regenerated plants increased 17-fold in anti-biotic-containing medium. Similar results wereobtained for cultured sorghum tissues [15]. The effectsof cefotaxime on cultured maize cells is not still stud-ied.

The objective of this work was to study the effectsof cefotaxime on callus induction and growth and alsoon morphogenesis in cultured maize tissues.

MATERIALS AND METHODS

Two maize (

Zea mays

L.) lines, A188 and R91, andtheir F1 hybrid were used. The R91 line was obtainedafter five cycles of self-pollination of plants regeneratedfrom callus tissues of the Chinese 31

×

Cateto SulinoGrosso F1 hybrid [16]. Seeds of the inbred A188 lineand of Chi 31

×

Cateto S.G. hybrid were kindly giftedby V.S. Shcherbak (Lukyanenko Research Institute ofAgriculture, Krasnodar).

The Stimulatory Effect of the Antibiotic Cefotaxime on Plant Regeneration in Maize Tissue Culture

S. A. Danilova and Yu. I. Dolgikh

Timiryazev Institute of Plant Physiology, Russian Academy of Sciences, Botanicheskaya ul. 35, Moscow, 127276 Russia;fax: 7 (095) 977-8018; e-mail: svdan@yandex.ru

Received May 14, 2003

Abstract

—In order to elucidate the effects of the antibiotic cefotaxime on callus growth and morphogenesis,we incubated embryogenic maize calli (

Zea mays

L.) of A188 and R91 lines and of their F1 hybrid with 50–500 mg/l cefotaxime throughout several subcultures. Cefotaxime did not affect the induction frequency andgrowth of the embryogenic callus but enhanced its morphogenesis. In both tested lines and a hybrid, the highestincrease in the number of regenerated plants was observed at the antibiotic concentration of 150 mg/l. Thedegree of morphogenesis stimulation and the range of cefotaxime concentrations effective in stimulation ofplant regeneration depended on the properties of calli obtained from tested genotypes.

Key words: Zea mays - cefotaxime - embryogenic callus - regeneration

METHODS

Abbreviation

: MS—Murashige and Skoog nutrient medium.

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DANILOVA, DOLGIKH

Plants were grown in a greenhouse in 5-l potspacked with soil at a temperature of 20

±

3

°

C under a16-h photoperiod. Pollination was performed undercontrolled conditions; male and female inflorescenceswere isolated.

Callus was obtained from immature embryos 1–2 mmin length excised on the 10–12th day after pollination.Prior to embryo excision, kernels were surface-steril-ized with the commercial bleach Belisna containingsodium hypochlorite (7–9% of active chloride) andwashed three times with sterile distilled water. For cal-lus induction and culturing, MS nutrient medium [17]supplemented with 1 mg/l 2,4-D was used. Subcultur-ing was performed every 20 days. Developing shootswere replanted to hormone-free MS medium. Tissueswere cultured and regenerants were grown at a temper-ature of 26

±

1

°

C, a humidity of 70%, an illuminance of1–2 klx, and a photoperiod of 16 h. Cefotaxime wasdissolved in sterile water and added to the nutrientmedium autoclaved and then cooled to 45

°

C immedi-ately before callus subculturing. Antibiotic concentra-tion in the medium varied from 50 to 500 mg/l. Cefo-taxime-devoid media were used as control ones. Theeffects of cefotaxime were evaluated at the stages ofcallus induction and growth and also during plantregeneration.

At least 50 inocula were analyzed in each treatment.Tables present means from three replications and theirstandard errors.

RESULTS AND DISCUSSION

When immature maize embryos were cultured onthe callus induction medium, along with callus forma-tion most embryos germinated. The antibiotic added atthis stage did not significantly affect the proportion ofimmature embryos producing embryogenic callus(Table 1) and the rate of callus growth. However, cefo-taxime enhanced substantially seedling development:independently of the antibiotic concentration, itincreased root and shoot lengths by three to five times.

To evaluate cefotaxime effects on callus growth andmorphogenesis, embryogenic tissues were cultured onantibiotic-containing medium throughout six subcul-tures. Cefotaxime did not accelerate callus growth: itsweight increased on the average fivefold on both antibi-otic-devoid and antibiotic-containing media. On theother hand, some cefotaxime concentrations stimulatedsubstantially the number of regenerated plants (Table 2).In the case of both lines tested and their

Ä188

×

R91

hybrid, cefotaxime was most effective at the concentra-tion of 150 mg/l. In A188 line, cefotaxime enhancedmorphogenesis only at the concentration of 150 mg/l,whereas, in

Ä188

×

R91

hybrid, the antibiotic concen-trations from 150 to 500 mg/l and in R91 line, the con-centrations from 100 to 500 mg/l were effective in acti-vation of regeneration. The degree of this activationdepended on the genotype. Thus, in A188 line, thenumber of shoots produced by a callus increased three-fold, in A188

×

R91 hybrid, 2.6-fold, and in R91 line,18-fold (Fig. 1).

Cefotaxime accelerated differentiation of maizeembryogenic callus. Antibiotic-induced stimulation ofplant regeneration was manifested starting from thesecond subculturing.

The number of regenerated plants attained the high-est values during the fifth subculture and then declinedsharply. When, whithout antibiotic, plant regenerationfrom the embryogenic callus lasted throughout twoyears, in the cefotaxime presence, callus differentiatedcompletely for six-to-seven subcultures (Fig. 2).

The grounds for growth and morphogenesis activa-tion by the antibiotic are not still elucidated. Two pos-sible causes are discussed in the literature: (1) somemicroorganisms are excluded from cultured tissues or(2) the products of antibiotic breakdown manifest bio-logical activity. The first supposition seems not veryprobable because many other antibiotics suppress cellgrowth and plant regeneration

in vitro

[18]. Thus, wedemonstrated that kanamycin completely suppressedmorphogenesis in maize calli at as a low concentrationas 25 mg/l (unpublished data).

One of the products of carbenicillin breakdown wasshown to be phenylacetic acid exhibiting auxin activity

Table 1.

The effect of cefotaxime on the frequency of em-bryogenic callus induction

Cefotaximeconcentration, mg/l

The proportion of embryosproducing embryogenic callus, %

A188 R91

0 52.6

±

4.7 82.5

±

7.1

100 48.3

±

3.3 76.8

±

6.4

150 54.5

±

4.2 80.3

±

7.8

250 56.2

±

5.1 79.6

±

5.9

500 51.4

±

4.9 84.2

±

8.1

Table 2.

The effect of cefotaxime on the frequency of plantregeneration

Cefotaxime concentration,

mg/l

The average numberof shoots produced by a single callus

A188 R91 A188

×

R91

0 1.2

±

0.3 0.3

±

0.1 1.3

±

0.2

50 1.2

±

0.2 0.5

±

0.1 1.3

±

0.3

100 1.5

±

0.3 2.5

±

0.3 1.6

±

0.4

150 2.4

±

0.2 5.4

±

0.5 3.4

±

0.3

250 1.3

±

0.3 3.5

±

0.4 2.3

±

0.3

500 0.8

±

0.4 2.9

±

0.5 2.1

±

0.3

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THE STIMULATORY EFFECT OF THE ANTIBIOTIC CEFOTAXIME 561

[19]. It might be that breakdown of cefotaxime, belong-ing to the same group of cephalosporins, also results inthe production of growth and morphogenesis stimula-tors.

The type of morphogenesis stimulation by the anti-biotic seems species-specific. Thus, in wheat culture,cefotaxime enhanced the frequency of embryogeniccallus formation from immature embryos and activatedtissue growth [12]; whereas, in maize culture, it did notaffect these processes (Table 1). In cultured sorghumtissues, cefotaxime prolonged the period of tissue capa-bility for plant regeneration [15]; whereas, in maizeculture, it acclerated callus differentiation. As distinctfrom other cereals, where the optimum antibiotic con-centrations for enhancement of growth and morpho-genesis were 50–60 mg/l, the higher cefotaxime con-centrations were required for improved maize plantregeneration (Table 2).

In maize culture, cefotaxime (100–250 mg/l) stimu-lated also the development of somatic embryoids pro-duced on the antibiotic-free medium and then trans-ferred to the regeneration medium containing cefo-taxime (Table 3). In this case, the optimum cefotaximeconcentration was also 150 mg/l: the proportion ofembryoids developed into plants was increased fivefoldin the A188 line and almost doubled in the

Ä188

×

R91

hybrid.The formation of somatic embryoids and their

development into plants are known to be controlled bydifferent genetic systems [20]. In our experiments,cefotaxime did not affect the production of embryo-genic callus (Table 1) but actively stimulated the devel-opment of preformed embryos (Table 3). It seems evi-dent that, in maize culture, the antibiotic was activeonly during the second stage of morphogenesis. Thiswould explain a difference in the morphogenesis stim-ulation in tested maize genotypes. The embryogeniccallus of the A188 line and

Ä188

×

R91

hybrid arecharacterized by spontaneous plant regeneration from

most somatic embryoids initiated in the callus tissue. Incontrast, the R91 line easily produces embryoids, sothat the whole callus surface is covered by green buds.However, their further development is blocked, and theregeneration frequency is very low. It might be that theantibiotic removed this block, inducing mass plantregeneration (Fig. 1). Therefore, the effect of cefo-taxime was most bright just in this maize line.

Our experiments permit a recommendation to treatembryogenic maize callus with the antibiotic cefo-taxime in order to improve plant regeneration. Alongwith the stimulation of morphogenesis, such a treat-ment accelerated callus differentiation, which permitsthe obtaining of a large number of plants for a shorttime-period (Fig. 2). A short period of tissue culturingwith a simultaneous increase in the number of regener-ated plants has evident advantages during plant genetic

1 2

Fig. 1.

The effect of cefotaxime on the development of the embryogenic callus of maize R91 line.(1) Cefotaxime-containing medium (150 mg/l); (2) Control medium.

1

5

10

2 3 4 5 6

Subculture number

15

20

25

30

35

40

0

% o

f to

tal r

eger

ants

Fig. 2.

The time-course of plant regeneration from embryo-genic callus of maize R91 line in cefotaxime-containingmedium (150 mg/l) throughout morphogenesis until com-plete callus differentiation.

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transformation and cell selection because reduces therisk of uncontrolled somaclonal variability [21].

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Table 3. The effect of cefotaxime on the developmentof regenerated plants

Cefotaximeconcentration, mg/l

The proportion of embryoidsdeveloped into plants, %

A188 A188 × R91

0 12.5 ± 4.5 44.2 ± 6.9

100 17.6 ± 5.4 54.5 ± 6.8

150 66.6 ± 6.5 91.2 ± 3.8

200 23.6 ± 5.8 80.8 ± 5.5

250 20.3 ± 5.3 54.0 ± 7.1