Plant Cell Rep (2005) 24: 556–560 DOI 10.1007/s00299-005-0942-z BIOTIC AND ABIOTIC STRESS Ramkumar Singh · Ravi K. Channappa · Farah Deeba · Nandi J. Nagaraj · Mohan K. Sukavaneaswaran · T. M. Manjunath Tolerance of Bt Corn (MON810) to Maize Stem Borer, Chilo partellus (LEPIDOPTERA: PYRALIDAE) Received: 9 November 2004 / Revised: 7 February 2005 / Accepted: 10 February 2005 / Published online: 12 June 2001 C Springer-Verlag 2005 Abstract Transgenic-corn plant (MON 810), expressing Introduction the Bt protein, Cry 1Ab, was evaluated under greenhouse conditions for its tolerance to the maize stem borer, Corn is one of the important cereal crops in the world Chilo partellus. Bt-corn (MON 810) provided effective (138.37 million hectares), with the largest area of 28.72 m protection against the stem borer even under high level hectare being in the USA. Among the genetically modified of larval infestation in the greenhouse. The observed commercially grown crops, Bt corn occupied the second tolerance has been viewed and discussed in the light place (9.8 m ha) after herbicide tolerant soybean (33.3 m ha) of susceptibility of C. partellus to Cry1Ab protein in and is cultivated in the USA, Canada, Argentina, South laboratory bioassays. Insect resistance management Africa, Spain and Germany (James 2002). Transgenic implications of the published tissue concentrations of corn expressing the Bt-gene Cry1Ab was developed in Cry1Ab in MON 810 and baseline-susceptibility recorded the USA for protection against the devastating European in the current study are also discussed corn borer, Ostrinia nubilalis (Lepidoptera: Pyralidae) and Keywords Chilo partellus . Susceptibility to Cry 1Ab . was approved for commercial cultivation in 1996 (James Screening of Bt-corn . Insect resistance management 1997). In India, corn is cultivated in over 6.51 m ha with Abbreviation more than 50% area in four states viz., Uttar Pradesh, Bt Bacillus thuringiensis Rajasthan, Madhya Pradesh and Bihar (Anon 1999). Insect m ha million hectare pests pose a challenge to higher productivity in corn in ppm parts per million (= micrograms per mL of diet in India. Among them, the stalk borers, viz., Chilo partellus bioassay) (Swinhoe) (Lepidoptera, Pyralidae) and Sesamia inferens (Walker) (Lepidoptera, Noctuidae) are the most serious. R. Singh · R. K. Channappa · F. Deeba · N. J. Nagaraj · The primary loss due to C. partellus infestation varied M. K. Sukavaneaswaran () · T. M. Manjunath from 26.7 to 80.4% in different agro-climatic regions of Monsanto Research Center #44/2A, Vasanth’s Business park, India (Chatterji et al. 1969). At the lower limit of 25%, the NH-7, Bellary Road, Hebbal, Bangalore, 560 092 India average loss in the Kharif growing season, at a conservative e-mail: [email protected] Tel.: +91-80-23622525 estimate, comes to Rs. 1105 million annually (Siddiqui and Fax: +91-80-23624343 Marwaha 1994). C. partellus also attacks sorghum, millets, rice, sugarcane, bajra and other grasses. The crop-loss Present address: in corn due to lepidopterans offers sufficient reason for R. Singh EG Technologies and Services Private Limited, 8-2-120/86/5, evaluating Bt-protected corn (maize) in India. Bt-corn Avenue:7 Road 3, Banjara Hills, Hyderabad, 34 India could provide scope for increasing maize production in e-mail: [email protected] India without resorting to increase in acreage or insecticide use. N. J. Nagaraj Department of Entomology, Kansas state University, Under tropical conditions, C. partellus completes ten to Manhattan, Kansas, 66506 4004 eleven generations in a year. The adults oviposit (in masses e-mail: [email protected] of 20–25 eggs each) on the undersurface of leaves at 2 to 3 leaf-stage of the plant. The emerging larvae suspend with T. M. Manjunath the help of silken thread and get dispersed to neighboring “Suma” 174 G Block, Sahakarnagar, Bangalore, 560 092 India plants by wind. Infested plants exhibit different symptoms e-mail: [email protected] during the various developmental stages. Damage to the 557 central growing shoot of young plants results in ‘dead- removal of insoluble material by centrifugation (8,000×g), heart’ symptoms. Plants that survive the initial attack show the resulting soluble Cry supernatant was adjusted to pH reduced inter-nodal length resulting in shoot ‘rosetting’. 8.5 and treated, when appropriate, with bovine pancreatic The young larvae also mine via the midrib into the stem trypsin (Sigma-Aldrich, St. Louis, MO), followed by dial- leading to reddening symptoms on the stem. In advanced ysis against 50 mM sodium chloride and 50 mM sodium stages of infestation, the third and later instar larvae tunnel carbonate, pH 10.5. The preparations were then further the stem while feeding, disrupting the transport of nutri- purified by anion-exchange chromatography using a Q- ents from the stem to the grains resulting in partially filled Sepharose column (Sigma-Aldrich, St. Louis, MO). Pro- grains. The larvae may also attack the developing cobs and tein concentrations were determined by densitometry using some times make galleries after feeding on the pith and bovine serum albumin as a standard. grains (Siddiqui and Marwaha 1994). While Bt corn has proved to be successful in protecting against European corn borer in the US and other countries, Diet the present greenhouse study was undertaken to evaluate if it could offer protection against the Indian stem borer, A general lepidopteran diet described by Nagarkatti and C. partellus, too. Seed materials were obtained from Mon- Sathyaprakash (1974) for Helicoverpa armigera was mod- santo Company, St. Louis, USA through National Bureau ified by the addition of 0.5% (5 g in a liter of diet) dried of Plant Genetic Resources (NBPGR), New Delhi, India. corn leaf powder and it was found to support growth and development of C. partellus for bioassays. Materials and methods Bioassay Partellus culture The insecticidal activity of Cry1Ab, against first instar lar- vae of C. partellus, was assayed using diet-incorporation A nucleus culture of C. partellus was collected from fod- method (Stone et al. 1989). Solubilised Cry1Ab was der maize fields in and around Bangalore, Karnataka, In- serially diluted to seven concentrations (1/4 or 1/3 of dia during February 2002. The field-collected larvae were the previous concentration) in sterile de-ionized water. A reared until pupation on cut pieces of whole baby corn. volume of 5.2 ml of diluted Cry1Ab standard, contained in Pupae were separated and placed in jars for moth emer- each of the serial dilution tubes, was thoroughly vortexed gence. A method for obtaining eggs in large numbers was ◦ with 20.8 ml of warm diet (50 C) and approximately 1 ml also standardized during the present study. Fifty freshly was poured in insect bioassay trays (CD International trays, emerged adults in equal sex ratio were released in a plastic Massachusetts, USA). Newly hatched, active larvae were jar (24×28 cm diameter) and its mouth covered with cloth transferred onto the solidified diet in the bioassay trays with the help of rubber bands. A polythene sheet was laid with a fine hairbrush (1 larva/well). After larval transfer, along inner wall of the jar and a few polythene ribbons bioassay trays were covered with self-adhesive pull-n-peel were also hung inside the jar from the cloth. These served tabs (CD International pull-n-peel tabs). The entire exper- as oviposition sites. Cotton wads soaked in 10% honey iment was done on a laminar flow clean air bench. The solution containing a diet supplement - Proteinex (Pfizer, ◦ trays were kept in an incubator maintained at 27±0.5 C. Mumbai) were placed in the jar as food for moths. Poly- Thirty-two larvae were used for each Cry1Ab concentra- thene strips and cotton wads were changed daily till the end tion and untreated control. The entire assay was repeated of the oviposition period. Oviposition jars were maintained ◦ 5 times. Observations on mortality and stadia of larvae in in growth chamber at 27 C70±5% relative humidity with each concentration were recorded when >90% of larvae a photoperiod of 16:8 (Light: Dark) hours. Eggs masses ◦ reached third instar in control (nine days after infestation). were kept in incubator (27 C) till larvae hatched out in two Observed mortality was corrected for mortality in control to three days. Neonates were immediately used for infest- treatments and lethal concentration with 95% fiducial ing bioassay trays or the experimental corn plants in the limits were calculated. Mortality related Lethal concentra- greenhouse. tions (LC50 and LC90); progress into II instar related moult inhibitory concentrations (MIC50 and MIC90); confidence limits and slopes for each assay were estimated using Susceptibility of C. partellus to Cry1Ab JMP 3.2.2 statistical package (SAS Institute 1989–1997). The various dose-response parameters were expressed as Preparation of Bt toxin micrograms of Cry1Ab per ml of diet (= parts per million, ppm). Cry1Ab toxin was prepared essentially as described by Brandt et al. 2003. Briefly, Cry1Ab toxin was prepared in a recombinant Bt strain based on EG10650, a deriva- Green-house evaluation of MON 810 tive of Bt strain EG10368 (U.S. Patent Nos. 5,759,538 and 5,962,264), and solubilized overnight at 4◦C in 100 mM The maize variety MON 810 genetically engineered sodium carbonate buffer, 10 mM DTT, pH 10.75. Following with the Bt gene of protein Cry 1Ab and its non-Bt 558 Table 1 Log dose response of LC 95% Fiducial limit LC 95% Fiducial limit Slope ± SE the neonates of Maize