Expression of Insect Resistance in in vitro-derived callus tissue infested with lepidopteran larvae

J. Plant Physiol.

Vol. 142. pp. 480-484 (1993)

Expression of Insect Resistance in in vitro-Derived Callus Tissue Infested with Lepidopteran Larvae BENJAMIN STEINITZ 1

1

*, AMOS NAVON2, MENACHEMJ. BERLINGER3, and MEIR KLEIN2

Department of Plant Genetics, 2 Department of Entomology, and 3 Gilat Regional Experimental Station, Agricultural Research Organization, The Volcani Center, Bet Dagan 50250, Israel

• (request for offprints) *' Contribution from the Agricultural Research Organization, The Volcani Center, Bet Dagan 50250, Israel. No. 3530-E,

1992 series Received April 22, 1993 . Accepted June 6,1993

Summary

Plant-insect relations were studied by rearing larvae of herbivorous lepidopteran insects on tissue culture-derived callus. The callus was generated from cotton (Gossypium hirsutum) and tomato (Lycopersion esculentum, L. chmielewskii and L. hirsutum) genotypes previously identified as differing in their insect field resistance. Calli were infested with newly hatched neonate larvae of Helicoverpa armigera, Spodoptera littoralis, Earias insulana and Phthonmaea operculella. Growth retardation of larvae and larval mortality were observed among insects fed with calli of resistant hosts. The response of the insects was very much dependent on exposure of the callus to light during its formation, and on the plant growth regulator composition of the callus growth medium. Our results indicate that factors imparting resistance in tissues of intact plants were also likely to be present in callus.

Key words: Gossypium hirsutum, Lycopersicon spp., Eanas insulana, Helicoverpa armigera, Phthonmaea operculella, Spodoptera littoralis, plant tissue culture, plant-insect interaction. Abbreviations: IAA = indole-3-acetic acid; NAA = O!-naphthaleneacetic acid; 2,4-D = 2,4-dichlorophenoxyacetic acid; 2iP = N 6-(2-isopentenyl)adenine; MS = Murashige & Skoog medium.

Introduction

Plant tissue cultures have been widely used in studying plant-microbe interactions and in screening genotypes for specific traits as a step in plant genetic manipulation programs (Daub, 1986; Jones, 1990; Van den Bulk, 1991). Thus far, screening for insect resistance with the aid of plant tissue cultures has been attempted with success in corn only. Callus cultures initiated from corn (Zea mays L.) hybrids with varying degrees of field insect resistance to the southwestern corn borer Diatraea grandiosella Dyar and to the fall armyworm Spodoptera /rugiperda E. Smith), were screened for expression of resistance to these herbivores. The extent of

a.

© 1993 by Gustav Fischer Verlag, Stuttgart

larval growth on callus correlated with the susceptibility of the plant from which the callus was derived (Williams et aI., 1983; Williams and Davis, 1985; Williams et aI., 1985). In similar experiments, Croughan and Quisenberry (1989) evaluated the utility of bermudagrass (Cynodon dacrylon (L.) Pers.) callus cultures in screening for fall armyworm resistance. Although differences among varieties in callus feeding experiments were observed, the specific resistance rating was not the same as that obtained in leaf screening experiments. The rationale for utilizing plant tissue cultures in screening plant germplasms for insect resistance is similar to that applied in screening for other traits. Plant tissue cultures can be raised continuously, under standardised conditions, and

Insect resistance in callus tissue

host tissue-insect interactions can be studied under a strictly controlled environment, thereby eliminating unpredictable factors, which often affect plant-insect field studies. While insect resistance based on physical defenses such as epidermal trichomes or cuticle texture cannot be evaluated (Southwood, 1986), these may mask additional defense components of secondary metabolites formed in subepidermal cells. Host callus-insect studies are a means to circumvent the epidermal barrier and thereby detect additional defense systems, which would otherwise remain unidentified in plantinsect assays. The present work was aimed at more critically investigating the utility of callus-insect systems for studying plant-insect relations. We describe the growth of newly hatched lepidopteran insect larvae reared on calli generated from cotton (Gossypium hirsutum L.) and tomato (Lycopersicum spp.) genotypes exhibiting a range of sensitivities against herbivorous insect pests in the field. In particular, we examine the extent of the expression of the insect resistance under specific tissue culture conditions. The study included four destructive pests, namely the African cotton bollworm Helicoverpa ar· migera (Hb.), the Egyptian cotton leafworm Spodoptera litto· ralis (Boisd.), the spiny bollworm Earias insulana (Boisd.), and the potato tuber moth Phthorimaea operculella (Zell.). The first two insects are feeders which cause substantial yield losses in major crops throughout most of the sub-tropical and tropical production areas of the world (Avidov and Harpaz, 1969). E. insulana is a cotton bollworm pest. The potato tuber moth is an oligophagous insect, its larvae feeding on potato, tomato and tobacco, mainly in warm temperate and sub-tropical areas (Fenemore, 1988).

Material and Methods

Plant material Cotton genotypes used were Gassypium hirsutum Acala SJ2, a commercial strain susceptible to herbivorous insects, and a primitive cotton accession V-64, for which Navon et a!. (1991) found a retarded development of H. armigera and S. littaralis insects feeding on its leaves. Seeds of both strains were obtained from the seed bank at the Dept. of Plant Introduction, A.R.O., Bet Dagan. The tomato species chosen for our study were a cultivated accession M-82 of Ly· copersicum esculentum Mill., susceptible to H. armigera and P. aper· culella larvea, and field resistant accessions of Lycapersicum chmie· lewskii Rick, Kes., Fob. and Holle (accession LA 1306) and Lycoper· sicum hirsutum Humb. and Bonp!. (accession LA 1777) Ouvik et a!., 1982). Tomato seeds were obtained from the Dept. of Plant Genet· ics, A.R.O. Cotton and tomato seeds were surface disinfected in 1 % sodium hypochlorite solution for 20 min, and then rinsed 3 times with sterilized distilled water. Seeds were germinated in 25 mm x 150 mm glass tubes sealed with polypropylene covers, in the dark for 4-6 days at 25° ± 1°C (tomato) or 30° ± 1°C (cotton), on a halfstrength MS basal medium salt (Murashige and Skoog, 1962) mixture (Sigma Co. USA), 20 g L -I sucrose, 2 g L - I Gelrite (Schweizerhall, NJ, USA), and 0.75 gL - I MgCb. Hypocotyl sections from etiolated seedlings served as explants for tissue culture initiation. In all experiments described below, we used a basal medium consisting of MS salts, plus (per liter) 100 mg myo-inositol, 0.5 mg nicotinic acid, 0.5 mg pyridoxine, 10.0 mg thiamine, 300 mg casein hy-

481

drolysate, 0.75 g MgCb and 2.0 g Gelrite. Sucrose and plant growth regulator composition are indicated in the particular experiments. Growth regulators were added to culture media either prior to autoclaving (NAA, 2,4-D, kinetin) or after autoclaving, as filter sterilized aqueous solutions (IAA, zeatin). The pH was adjusted to 5.8 with NaOH before addition of Gelrite, and the medium was autoclaved at 1.2 Kg - 1 em - 2 for 20 min at 121°C. Cotton callus was initiated in 9 Xcm Petri dishes with 25 mL of a basal medium supplemented with (per liter) 30g glucose, 0.1 mg 2,4-D and 0.1 mg kinetin (Zimmerman and Robacker, 1988). Calli were routinely subcultured every 10 -14 days on the same medium, and the cultures were incubated in a Percival E-30B growth chamber at 30° ± 1 °C either in the dark or under continuous white fluorescent light of 80!Lmolm-2sec-1 at shelf level, provided by General Electric F24T12-CW-HO cool white fluorescent tubes. Tomato callus was initiated in Petri dishes with 25 mL of basal medium supplemented with (per liter) 30 g sucrose, 2 mg IAA and 0.2 mg zeatin. Two weeks after culture initiation, calli were transferred and maintained on a medium having one of the following auxin and cytokinin combinations (per liter): 4 mg NAA and 1 mg kinetin, 1 mg NAA and 10 mg 2iP, 2 mg IAA and 1 mg kinetin. All cultures were routinely subcultured every 7 days. Callus cultures were grown in a culture room at 25° ± 1°C under a photoperiod of 16 h light and 8 h dark. Fourty !Lmol m -2 sec- I white light at shelf level was provided by Sylvania XL F40 cool white fluorescent tubes.

Insect biaassay Details of the culture conditions for raising insect colonies of the African cotton bollworm H. armigera were described by Navon et al. (1990). S. littaralis was reared on castor beans, the potato tuber moth P. aperculella on potato tubers, and E. insulana on a diet described by Klein et al. (1981). All following steps were carried out under aseptic conditions in a laminar air-flow bench. Insect eggs were surface disinfected in 1.8 % v/v formaldehyde for 60 min, rinsed 3 times in distilled water, blotted dry and placed in a Petri dish. The eggs were kept until hatching in dry Petri dishes in a dark incubator at 25° ± 1°C, and the subsequent neonate bioassay was based on the procedures of Navon et a!. (1990). A plastic grid with 211 cm 3-cells was placed in a 9em-Petri dish (see Navon et al., 1990, Fig. 1). Plant tissue was removed from the culture medium, blotted dry from free fluids on a tissue paper, and each grid cell was packed with callus. A single neonate larva was placed in each of the grid cells, the grid was covered with a What man No. 1 filter paper disc, and a second plastic grid, overlapping the first one, was placed on top of the filter paper. The Petri dish lid was pressed against the grids with the aid of rubber bands and the dish was sealed with Parafilm. The insect- inoculated calli were incubated in plant tissue culture growth chambers with relative humidity kept above 90 %. The rearing period of the neonate larvae was 6 days for H. armigera, S. littaralis and E. insulana, and 8 days for P. aperculella. Calli retained their viability under the conditions described. Neonate weight at onset of the tests was about 50 !Lg for P. aperculella and approximately lOO!Lg for the other insects. By the end of the rearing period each larva was weighed separately, and the mean larval weight per treatment was calculated. Larval death was also scored. Fifteen larvae were used for each treatment in an experiment. Ex· periments were repeated four or five times at different dates. The data represent the mean value of these replica and were subjected to analysis of variance.

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BENJAMIN STEINITZ, AMos NAVON, MENACHEMJ. BERLINGER, and MEIR KLEIN

Results

which calli were cultured, and the resulting metabolic response correlated to pigmentation, affected subsequent insect development.

Cotton Callus was initiated in the dark from hypocotyl segments of dark-germinated seedlings. One month after callus initiation calli were divided and subcultured in either the dark or in c~ntinuous light. Exposure of the callus to light induced variegation into green or red-purple pigmented cells, the latter probably reflecting synthesis and. accumulat~on of s~c­ ondary metabolites such as anthocyanms and tamns ..For msect feeding experiments, we sorted three types of callI, based on colour: (dark grown) white, and (light grown) green or purple. Neonate larvae of H armigera, S. littoralis, and E. insulana were placed on the three callus types of each cott?n genotype, and their growth (fresh weIght) and mortalIty was recorded. Insect weight was affected by cotton genotype (Table 1). A small retardation of growth of H armigera was seen in larvae fed with white callus of cotton strain V-64. E. insulana showed no significant growth retardation on white or green V-64 callus, but a significant growth inhibition on purple V64 callus, compared to larval growth on purple Acla 5J2 c.alIus. For S. littoralis, a larval weight difference between msects grown on the two cotton genotypes was evident on white and green calli, but not on the purple callus. The size of the insect population used throughout the present study was too small to allow a statistical assessment. of mortality among the larvae, however, percentage mortalIty over the total number of insects subjected to a treatment, IS indicated in Table 1. Neonate mortality appeared in E. insu· lana reared on purple V-64 callus, and in S. littoralis reared on green and purple V-64 calli. . Within a given cotton genotype, the larvae of all three ~n­ sect species had the highest weight when reared on the wh~te callus, the smallest weight on the purple callus, an~ an mtermediate weight when fed with the green cal~us. EVI~entlr, in addition to the host genotype effect, the lIght regIme m

Table 1 : Mean larval weight (mg ± S.E. of the mean) and mortallity percentage of Helicoverpa armigera,. Earias insulana and Spodoptera littoralis larvae reared on cotton call!. Callus pigmentation White

Green

H armigera

Purple

Cotton genotype Acala SJ2 [S]1 V-64 [Rr

14.0 ± 3.5a2 8.3±2Ab

Acala SJ2 [S] V-64 [R]

20.6 ± 5.3 a 17.6 ± 8.3 a 17.2±4.6a 11.0± 4.1a

Acala SJ2 [S] V-64 [R]

43.8 ± 6.2a 38.3 ± 10Aa 15.3 ± 1.2a 30.3±5.5b 18.8± 4.9b(3) 13.3±4.1a(10}

9.3 ± 0.9a 7.8± 1.8a

804 ± 1.5a 5.9 ± 1.8a

E. insulana 15.9 ± 6.1 a 8.2 ± 4.8 b (9}3

S. littoralis

1 [S], [R]: Susceptible and resistant genotypes, respectively, as defined on field grown plants.. .. 2 Whithin a given insect species and callus pigmentatIOn, means ~e separated by Duncan's multiple range test; the same letters deSignate non significant difference at p < 0.05. . 3 Where mortality occurred, this is shown as percentage In parenthesis.

Tomato Calli were maintained on three media differing in their auxin and cytokinin composition. With these media it was possible to generate, in each tomato species, calli o.f significantly different visual (intensity of green colouratlOn) and mechanical (firm/soft) properties. Feeding experiments were conducted with the cotton bollworm and the potato tubermoth larvae (Table 2). L ycopersicon hirsutum developed a very soft and watery callus on media with NAA and either kinetin or 2iP. During preliminary experiments neonate larvea «drowned» in such calli and we concluded that tissue which is too soft does not suit 'insect feeding experiments. Therefore, Table 2 does not include data on larval development for insects placed on such calli. Helicoverpa armigera neonates, when reared on calli formed on a medium with NAA and 2iP, grew equally well on tissues of L. esculentum and L. chmielewskii. However, P. operculella neonate larvae reared on L. esculentum tissue reached a weight that was more than double the larval weight when reared on L. chmielewskii callus, and mortality occurred only among these larvae fed with L. chmielewskii callus. A different result emerged with calli generated on a medium with NAA and kinetin; H armigera grew on L. esculentum slightly faster then on L. chmielewskii callus, but P. operculella larvae developed equally well on. both tomato species. Finally, larval development of both msects was assayed on calli of the three tomato species regenerated on a medium with IAA and kinetin. Helicoverpa armigera and P. operculella neonates grew b~st on L. esc~lentum ~allus, grew least on L. hirsutum and displayed an mtermedlate growth rate on L. chmielewskii (Table 2). Insect mortality was observed among P. operculella neonates only upon feeding with

Table 2: Mean larval weight (mg ± S.E. of the mean) and mortality percentage of Helicoverpa armigera and Phthorimaea operculella larvae reared on calli of three Lycopersicon species. Medium [auxin/cytokinin]

Lycopersicon spp. L. esculentum [5]1 L. chmielewskii [R]1 L. hirsutum [R] L. esculentum [5] L. chmielewskii [R] L. hirsutum [R]

NAA/2iP

NAA/kinetin

14.0 ± 4.0a2 12.S ± 7.0a

22.S ± 6.2a 13.S ± 4.2a

4.S ± 1.3 a 1.9 ± OAb (10)3

H armigera

P. operculella

5.6 ± 1.Sa 404 ± 0.4a (S)

IAA/kinetin

27.7 ± 3.0a 14.0±4.0b S.2 ± 2.Sc 12.0 ± 1.7 a 7.3± 1.Sb 2.S ± 2.0c (lS)

1 [S], [R]: Susceptible and resistant genotypes, respectively, as defined on field grown plants. ., . . 2 Whithin a given insect species and an auxin/cytokinin combination, means are separated by Duncan's multiple range test; the same letters designate non significant difference at p < 0.?5. 3 Where mortality occurred, this is shown as percentage In parenthesis.

Insect resistance in callus tissue

L. chmielewskii calli generated on media supplemented with NAA and either 2iP or kinetin, or with L. hirsutum callus formed on medium with IAA and kinetin. Larval death did not appear in insects fed with L. esculentum callus. In summary, neonate larvea growth and development was host genotype-dependent, and influenced by the effect of the growth regulators in the medium on the callus.

Discussion Callus formation can be induced in a wide range of plant species and, as our work demonstrates, callus-insect resistance assays can be successfully run, even with plants recalcitrant to regeneration such as Gossypium spp. (Trolinder and Xhixain, 1989; Gould et a1., 1991). Herbivorous insect resistance in cotton is often based on the production of secondary metabolites such as anthocyanin, flavonoids, tannins and gossypol (Hedin et al., 1983; Hedin et a1., 1991). Gossypol synthesis in cotton cell cultures was described by Heinstein and EI-Shagi (1981), and light has been found to stimulate the formation of a wide range of secondary compounds in cell cultures (Seibert and Kadkade, 1980; Seitz and Hinderer, 1988; Zaprometov, 1988). It is, therefore, not unexpected that light exposure of cotton tissue cultures would have consequences on the development of larvae feeding on such calli, as shown in Table 1. The factors detrimental to larval development in insects reared on tomato callus (Table 2) are at present not known. We assume, based on work by Fery and Kennedy (1987) and Barbour and Kennedy (1991), responses are due to secondary metabolites the levels of which might be governed by plant growth regulators in culture medium (Kudakasseril and Staba, 1988; Seitz and Hinderer, 1988). Plant growth regulator-dependent alterations in in vitro expression of resistance have been described for other host plant-pathogen interactions (e.g. Haberlach et a1., 1978; Huettel and Hammerschlag, 1986; Ashikawa et a1., 1991). In the course of our work we encountered two difficulties in the callus-insect assays: 1) A host callus with mechanical rigidity sufficient to support the moving larvae is required, yet a vigorous growth of the tissue is frequently associated with the generation of a soft callus. 2) Calli consist of heterogeneous cell populations and consequently a heterogeneity with regard to insect resistance may appear (Table 1). This heterogeneity can lead to large fluctuations in the results and, in extreme cases, could hamper distinction between sensitive and resistant plant genotypes. Several conclusions can be drawn from our observations. Firstly, factors present in field grown host plants, which imparted insect resistance, were likely to be present in callus also. Obviously, we deal with chemical resistance factor(s) with expression independent of the differentiation of organized tissues and organs. Secondly, with larval rearing on in vitro-derived callus, the study of plant-insect interactions can be expanded not only to different host plant genotypes but also to different insect species. Thirdly, in cotton and tomato, the discrimination between genotypes differing in their field insect-resistance by use of a plant tissue cultureinsect assay is feasible, provided calli express the resistance factors. Our results indicate that host tissue incubated in

483

unappropriate conditions may loose a part or the entire capability to form the resistance factors. Further development and rigorous standardization of the procedures are essential before screening with different species of plants and herbivorous insects can be implemented. Once refind, the in vitro test might be considerably less elaborate than current greenhouse or field tests. Acknowledgements

We thank Dr. M. Pilowski of the Dept. of Plant Genetics, A.R.O., and Mr. M. Zur of the Dept. of Introduction, A.R.O. for providing tomato and cotton seeds, respectively. We express our appreciation for the skillful technical assistance of Mrs. A. Ben Meir and Mrs. Sara Keren. Thanks are due also to Drs. V. Gaba, M. Koch and A. Zelcer for helpful comments on the manuscript.

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