Molecular Breeding 4: 33±37, 1998. 33

c 1998 Kluwer Academic Publishers. Printed in Belgium.

Insect-resistant transgenic brinjal plants

Polumetla A. Kumar, Ajin Mandaokar, Karra Sreenivasu, Swarup K. Chakrabarti,

Suman Bisaria, Surat R. Sharma, Sarvjeet Kaur & Rameshwar P. Sharma National Research Centre for Plant Biotechnology, Indian Agricultural Research Institute, New Delhi-110012,

India ( author for correspondence)

Received 21 April 1997; accepted in revised form 21 October 1997

Key words: Bacillus thuringiensis, insecticidal crystal protein gene, cry1Ab, brinjal, fruit borer, orbonalis, transgenic plants

Abstract

A synthetic cry1Ab gene coding for an insecticidal crystal protein (ICP) of Bacillus thuringiensis (Bt) was transferred to brinjal () by cocultivating cotyledonary explants with Agrobacterium tumefaciens. Transformant plants resistant to kanamycin were regenerated. Hybridization experiments demonstrated gene integration and mRNA expression. Double-antibody sandwich ELISA analysis revealed Bt toxin protein expression in the transgenic plants. The expression resulted in a signi®cant insecticidal activity of transgenic brinjal fruits against the larvae of fruit borer (Leucinodes orbonalis). The results also demonstrated that a synthetic gene based on monocot codon usage can be expressed in dicotyledonous plants for control.

Introduction growing fruits. The larvae, upon hatching, bore into the fruits and shoots causing severe damage and rendering Bacillus thuringiensis (Bt), a gram-positive, spore- the fruits inedible. In the present study, we transformed forming bacterium, produces a variety of insecticidal brinjal plants using a lepidopteran-speci®c, synthet- crystal proteins (ICP) toxic to lepidopteran [13]. ic cry1Ab gene extensively modi®ed for high expres- The ICP genes of Bt have been successfully engin- sion in plant cells. The transgenic brinjal fruits were eered into many crop plants to yield resistance against demonstrated to be signi®cantly resistant to the larvae lepidopteran insects [11]. The levels of toxin expres- of L. orbonalis. sion in plants, however, have been insuf®cient when the native genes were used, necessitating the use of a truncated version of the genes and modi®cation of the Materials and methods coding sequence, such as removal of potential RNA processing and polyadenylation signals and optimiza- Plasmids and bacterial strains tion of the codon usage to achieve higher expression of the gene in plants [14]. When expressed in plant Plasmids pBT 1291 [5] and pBinAR [7] and derivatives cells these modi®ed genes conferred signi®cant protec- were transformed into Escherichia coli strain XL-1 tion against insects to important crops such as cotton, Blue (Stratagene). For plant transformation, the bin- maize, rice, and [11, 13]. ary vector was mobilized into A. tumefaciens EHA105 Brinjal (eggplant) is one of the most important (pEHA105) [8]. vegetable crops in , especially the Indian sub- continent where the diet is predominantly vegetarian Reagents and enzymes in nature. Brinjal is extensively damaged by the lepid- opteran insect Leucinodes orbonalis which is speci®c Restriction endonucleases and T4-DNA ligase were to this crop [1]. The adults lay eggs on the buds and purchased from Stratagene. GeneScreen Plus nylon 34 membranes were procured from NEN Research Enzyme-linked immunosorbent assay (ELISA) Products (Dupont). All reagents and chemicals used were of molecular biology grade (Sigma). Rapidly growing leaves were frozen and ground in liquid nitrogen. The leaf powder was extracted with Construction of plant transformation vector phosphate-buffered saline/Tween (PBST). The leaf extracts were centrifuged and the supernatants were Plasmid pBT 1291 with a synthetic cry1Ab gene [5] immediately used for protein estimation [2]. The was restricted by XbaI and the 2.4 kb fragment with extracts were tested by a double-antibody sandwich the gene was inserted into the XbaI site of the binary quantitative ELISA [3]. Antibodies raised in rabbits vector plasmid pBinAR [7] which contains a CaMV against pure Cry1Ab protein (Prof. Donald Dean, Ohio 35S promoter and octopine synthase poly(A) sequence. State University, Columbus, OH) were used. Goat anti- rabbit (GAR) IgG conjugated with horseradish per- Plant transformation oxidase (HRP) was used as secondary antibody. O- phenylenediamine dihydrochloride (OPD) was used as The plasmid pBinBt was transferred into A. tumefa- substrate and the orange-brown colour was detected ciens strain EHA105 (pEHA105) by the freeze-thaw by the ELISA plate reader at 492 nm. Both primary transformation method [6]. Brinjal (Solanum melon- antibody and GAR-IgG-HRP conjugate were used at gena cv. Pusa Purple Long) seedlings were raised 1:1000 dilution with phosphate-buffered saline (PBS). aseptically on half-strength Murashige and Skoog The levels of Cry1Ab protein in the leaf extracts were (MS) medium. Cotyledonary leaves from 12-day old determined by extrapolation to a standard curve based seedlings were excised and used for infection and on sample and standard absorbance values. cocultivation with A. tumefaciens [16]. The explants were incubated on regeneration/selection medium con- Insect culture and bioassays taining MS salts, B5 vitamins, 3% sucrose, 1.0 mg/l zeatin, 100 mg/l kanamycin, 500 mg/l cefotaxime and Damaged fruits of brinjal were collected from the farm- 0.2% Phytagel (pH 5.8). The regenerated shoots were ers' ®elds around New Delhi. The fruits were placed grown on root induction medium containing MS salts, in large glass jars in a culture room (28  C, 60% relat- 3% sucrose, 50 mg/l kanamycin, 300 mg/l cefotaxime ive humidity). The fully grown larvae came out of the and 0.2% Phytagel. The rooted plants were trans- fruits and pupated. The adult moths were collected and planted into small pots containing vermiculite. After transferred to jars containing cotton swabs dipped in establishment, the plants were shifted to large earthen honey. The eggs were laid in patches and larval emer- pots in the greenhouse. gence occurred after 4 days. Four neonate larvae per fruit were placed on rapidly growing fruits of control Southern and RNA blot analysis and transgenic plants. The fruits were sliced open after 9 days to assess the damage as measured by the num- Isolation of total genomic DNA from the leaves was ber and weight of larvae that could establish residence. performed by the procedure described by Saghai- The bioassays were repeated twice. Maroof et al. [17]. DNAs were restricted with XbaI, electrophoresed on 0.8% agarose gel and transferred onto GeneScreen Plus membrane. Southern hybridiz- Results and discussion ation was carried out with a 32P-dCTP-labelled XbaI insert (2.4 kb) separated from pBT 1291 according The ICP of B. thuringiensis, Cry1Ab, is toxic to a to Sambrook et al. [18]. Total RNA was isolated broad range of lepidopteran insects [11]. A synthetic from young and mature leaf tissues with guanidine cry1Ab gene modi®ed for plant codon usage and car- thiocyanate-based RNeasy Plant Total RNA Kit (Qia- rying a castorbean catalase intron was cloned into a gen) according to the manufacturer's instructions. The binary Agrobacterium vector, pBinAR [7]. The chi-

RNAs (2.5, 5, 10 and 20 g) were dot-blotted on a meric gene cassette in pBinBt consisted of the CaMV

GeneScreen Plus membrane and the membrane was 35S promoter, a synthetic cry1Ab gene and the 30 end hybridized with the 32P-labelled XbaI fragment of pBT of the octopine synthase gene from the Ti plasmid 1291. of Agrobacterium (Figure 1). A. tumefaciens strain EHA 105 was used to transform brinjal cotyledon- 35

Figure 2. Southern hybridization of ICP gene in transgenic brinjal Figure 1. Plant transformation vector pBinBt containing the ICP plants. Total DNA was isolated from the transformed and control gene, cry1Ab; NptII, neomycin phosphotransferase II; LB, left bor- brinjal plants and fully digested with XbaI. The hybridization was der; RB, right border; pAOCS, terminator of octopine synthase gene. performed with radiolabelled XbaI fragment from pBT 1291 (2.4 kb) as probe. Lane P, pBT 1291 DNA; lane C, normal brinjal plant; lanes 1±6, transgenic brinjal plants (BT1, BT2, BT3, BT4, BT5 and BT6, respectively). The upper band in lane P is undigested DNA. ary explants. Thirteen plantlets resistant to kanamy-

cin were regenerated. Total genomic DNA (10 g) from each plant was loaded onto a nylon membrane rabbit antibodies speci®c for Cry1Ab protein to detect using a dot blot apparatus (BioRad). The membrane the levels of toxin expression in the transgenic plants. was hybridized with the radiolabelled XbaI insert Toxin levels as measured by ELISA were converted to of pBT1291. The analysis revealed that only six of ng toxin per milligram total soluble protein (Figure 4). the thirteen putative transformants were positive (res- Four plants out of the six transformants tested exhibited ults not shown). The genomic DNAs from these six detectable levels of Cry1Ab protein (125±142 ng per plants were restricted with XbaI and separated on a milligram soluble protein). The maximum amount of 0.8% agarose gel. The DNAs were transferred onto a Cry1Ab protein found in BT6 corresponded to about nylon membrane and hybridized with the radiolabelled 0.02% of total soluble protein. The results from the gene insert. The results clearly demonstrated trans- insect bioassay revealed that the fruits from the plant gene integration into the genomic DNA (Figure 2). BT6 were completely free from the damage and the lar- The normal brinjal plant DNA (Figure 2, lane C) did vae could not establish residence (®gure 5). Fruits from not show any hybridization signal as expected. Total the other plants exhibited partial resistance as shown RNAs from four of the six transformant plants (BT 2, in Figure 4. The larvae that could survive decreased in 3, 4 and 6) were dot-blotted and hybridized with the weight (40%±88%) relative to that of control larvae. labelled gene insert. Figure 3 shows that all four plants It has been convincingly demonstrated by sever- expressed cry1Ab-speci®c mRNA. Plant BT4 exhib- al groups [4, 5, 9, 10, 15, 19] that modi®cations at ited the highest level of RNA expression. The levels the nucleotide level of prokaryotic genes can signi- of cry1Ab mRNA were higher in the young leaves of ®cantly enhance expression of bacterial transgenes in the plants (BT 3, 4 and 6) than in those in the mature higher plants. Insecticidal crystal protein (ICP) genes leaves and reverse was the case for plant BT 2. Prob- of B. thuringiensis were extensively modi®ed keep- ably factors other than the age of the leaf also in¯uence ing in view the codon usage of higher plants [15], the levels of RNA expression. dicotyledonous plants [9] or monocotyledonous plants Double antibody sandwich ELISA analysis was [5, 10] for better expression in the respective crop performed using immunoaf®nity-puri®ed polyclonal species. When compared with the transgenic plants 36 expressing wild-type genes, plants carrying modi®ed genes express the Bt toxin manifold and suf®cient enough to achieve complete control of lepidopteran and coleopteran pests [11, 13]. We have generated six transgenic brinjal plants, through an A. tumefa- ciens approach using a cry1Ab gene completely mod- i®ed for rice codon usage [5]. The transgenic plants expressed the cry1Ab gene constitutively under the control of CaMV 35S promoter. The expression was suf®cient enough to confer resistance to the lepidop- teran fruit borer L. orbonalis. Molecular analysis of the plants revealed transgene integration and mRNA Figure 3. RNA blot analysis of cry1Ab mRNA in transgenic plants. Total RNA was isolated from young (Y) and mature (M) leaves of expression. ELISA experiments demonstrated that the transgenic brinjal plants and dot-blotted onto a GeneScreen mem- protein levels in the transgenic plant leaves are com- brane. The membrane was hybridized with radiolabelled 2.4 kb XbaI parable to the levels obtained in other transgenic crop fragment from pBT1291. C, control brinjal plant; BT2, BT3, BT4 plants expressing synthetic Bt genes [5, 10]. We have and BT6, transgenic brinjal plants. used a fully modi®ed cry1Ab gene designed for use in

rice [5]. The overall G +C content of the modi®ed gene

is 59.2% which is higher than the G+C contents of the genes from dicotyledonous plants. However, this did not seem to impose any limitation in expressing the gene in brinjal, a member of the family Solanaceae. The levels of Cry1Ab expressed in the leaves of four of the six transgenic plants were found to be simil- ar. However, there were signi®cant differences in the insect mortality as recorded by the fruit bioassays. The results suggest that the levels of the toxin in the fruits were probably not high enough to confer complete protection against L. orbonalis. Hence, it would be Figure 4. Expression of the Cry1Ab protein in transgenic brinjal desirable to use an overexpressing fruit-speci®c pro- plants (open bars) and larval mortality (closed bars). The amount moter for better expression of the cry1Ab gene in the of Cry1Ab protein was detected using a double sandwich ELISA fruit tissues. It has been advocated that the expression technique. Insect bioassays were done as described in Materials and methods. The error bars represent standard error of the mean. of Bt ICPs at `high doses' will confer complete protec- tion to the plants and also prevent the development of resistance in the insects [13]. Glasshouse tests and ®eld evaluation are necessary to critically evaluate the usefulness of these transgen- ic brinjal plant vis-a-visÁ L. orbonalis infestation and damage. Experiments are in progress to investigate the gene copy integration and segregational analysis in T1 generation.

Acknowledgements

We thank Drs Raj Bhatnagar and V. S. Reddy of ICGEB, New Delhi, for their help during molecular analysis and Dr K.S. Murty of NCIPM for help in the bioassays. Figure 5. Bioassay results with Leucinodes orbonalis larvae of control (C) and transgenic (6) brinjal fruits. 37

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