Development and bioassay of Cry1Ac-transgenic eggplant (Solanum melongena L.) resistant to shoot and fruit borer By J. K. PAL 1 , M. SINGH 1 , M. RAI 1 , S. SATPATHY 1 , D.V. SINGH 2 and S. KUMAR 3 * 1 Indian Institute of Vegetable Research, P.O. Box 5002,Varanasi-221005, India 2 Department of Botany, Udai Pratap Autonomous College,Varanasi-221002, India 3 AVRDC-ICRISAT Project, International Crops Research Institute for the Semi-Arid Tropics, BP- 12404, Niamey, Niger (Via-Paris, France) (e-mail: s.kumar.avrdc@cgiar.org) (Accepted 24 February 2009) SUMMARY Eggplant (Solanum melongena L.) is an important fruit vegetable, commercially cultivated in the tropics and sub- tropics. However, the most serious constraint on eggplant production is damage caused by eggplant shoot and fruit borer (ESFB).The development of resistant transgenic plants, using an insecticidal crystal protein gene, is an available crop protection option. A Cry1Ac gene obtained from the National Research Center for Plant Biotechnology (NRCPB), New Delhi, India, was transformed via Agrobacterium-mediated gene transfer into an improved inbred line of eggplant (IVBL-9). Hypocotyls proved to be the most effective and suitable explants, with a transformation frequency of 17.3% and 2.9 shoots per explant. PCR and Southern blot analyses confirmed the presence of a single copy insertion of the Cry1Ac gene in seven independent transgenic plants. The insertion of a single copy of the gene was also confirmed by segregation analysis of T 1 seed from T 0 plants. ELISA analyses revealed the presence of the Cry1Ac protein, and quantitative estimates confirmed significant levels of Cry1Ac protein (2.46 – 4.33 ng ml –1 leaf extract) expressed in all seven transformed plants. High levels of expression of this insecticidal protein resulted in significant larval mortality and in the reduced growth of any surviving larva on transformed eggplant tissue. E ggplant (brinjal, aubergine or guinea squash; Solanum melongena L.) originated in India (Gleddie et al., 1986) and is an important fruit vegetable in both tropical and temperate regions. Eggplants are a good source of vitamins and minerals (particularly iron), providing a nutritional value comparable to that of tomato (Kalloo, 1993). Eggplants are also used for the treatment of asthma, bronchitis, and cholera, and consumption of the leaves is beneficial for lowering blood cholesterol (Khan, 1979). In the tropics and sub- tropics, the major limitation on eggplant production is severe damage (up to 60% loss) caused by a Lepidopteran insect pest, the eggplant shoot and fruit borer (ESFB; Leucinodes orbonalis; Mall et al., 1992). The larvae of ESFB start by damaging the shoots of young plants, and later damage (bore) into the fruit, making them unmarketable. Control measures for this pest rely mainly on the application of insecticides, which are often ineffective because the insect tends to develop resistance and those larvae already inside the fruit escape contact with the insecticide. The indiscriminate use of insecticides has also led to increased costs of production and possible environmental hazards. The lack of any natural source of resistance to ESFB in all cultivated and conventionally cross-compatible species of Solanum has been a major obstacle to developing ESFB-resistant cultivars. Plant genetic engineering (Vaeck et al., 1987) using insecticidal crystal proteins (ICPs; -endotoxins or Cry proteins) from strains of Bacillus thuringiensis has permitted the development of transgenic eggplants resistant to ESFB. Guri and Sink (1988) first reported the transformation of eggplant using Agrobacterium tumefaciens, followed by others (Fillippone and Lurquin, 1986; Rotino and Gleddie, 1990; Hanyu et al., 1999). Transgenic eggplants resistant to ESFB by virtue of possessing Cry gene constructs have been developed in India (Kumar et al., 1998). Five hybrids derived from transgenic EFSB- resistant inbred lines were developed by the Maharashtra Hybrid Seeds Company (Mahyco, Jalna, India) and are at an advanced stage of multi-site field trials. Considering the wide range of marketable types of eggplants based on fruit colour, shape, size, the presence or absence of spines on the calyx, and the need for open- pollinated resistant transgenic lines, efforts were made to develop ESFB-resistant transgenic lines in genetic backgrounds of the most desirable market types. The results of one such event are described in this paper. MATERIALS AND METHODS Culture conditions Agrobacterium tumefaciens strain LBA4404 harbouring the binary vector pBinAR (Hofgen and Willmitzer, 1990) carrying the neomycin phosphotransferase (npt-II) gene with a nopaline synthase (NOS) promoter and a Cry1Ac gene fused to a well-known constitutive plant promoter (CaMV35S) and *Author for correspondence. Journal of Horticultural Science & Biotechnology (2009) 84 (4) 434–438