Monika Singh

Bioinformatic analysis for allergenicity assessment of Bacillus thuringiensis Cry proteins expressed in insect-resistant food crops

The novel proteins introduced into the genetically modified (GM) crops need to be evaluated for the potential allergenicity before their introduction into the food chain to address the safety concerns of consumers. At present, there is no single definitive test that can be relied upon to predict allergic response in humans to a new protein; hence a composite approach to allergic response prediction is described in this study. The present study reports on the evaluation of the Cry proteins, encoded by cry1Ac, cry1Ab, cry2Ab, cry1Ca, cry1Fa/cry1Ca hybrid, being expressed in Bt food crops that are under field trials in India, for potential allergenic cross-reactivity using bioinformatics search tools. The sequence identity of amino acids was analyzed using FASTA3 of AllergenOnline version 10.0 and BLASTX of NCBI Entrez to identify any potential sequence matches to allergen proteins. As a step further in the detection of allergens, an independent database of domains in the allergens available in the AllergenOnline database was also developed. The results indicated no significant alignment and similarity of Cry proteins at domain level with any of the known allergens revealing that there is no potential risk of allergenic cross-reactivity.

Decaplex and real-time PCR based detection of MON531 and MON15985 Bt cotton events.

The genetically modified (GM) Bt crops expressing delta-endotoxins from Bacillus thuringiensis provide protection against a wide range of lepidopteron insect pests throughout the growing season of the plant. Bt cotton is the only commercialized crop in India that is planted on an area of 7.6 million hectares. With the increase in development and commercialization of transgenic crops, it is necessary to develop appropriate qualitative and quantitative methods for detection of different transgenic events. The present study reports on the development of a decaplex polymerase chain reaction (PCR) assay for simultaneous detection of transgene sequences, specific transgene constructs, and endogenous stearoyl acyl desaturase (Sad1) gene in two events of Bt cotton, i.e., MON531 and MON15985. The decaplex PCR assay is an efficient tool to identify and discriminate the two major commercialized events of Bt cotton, i.e., MON531 and MON15985, in India. Real-time PCR assays were also developed for quantification of cry1Ac and cry2Ab genes being employed in these two events. The quantitative method was developed using seven serial dilutions containing different levels of Bt cotton DNA mixed with a non-Bt counterpart ranging from 0.01 to 100%. The results revealed that the biases from the true value and the relative standard deviations were all within the range of ±20%. The limit of quantification (LOQ) of the developed real-time PCR method has also been established up to 0.01%.

Multitarget Real-Time PCR-Based System: Monitoring for Unauthorized Genetically Modified Events in India

A multitarget TaqMan real-time PCR (RTi-PCR) based system was developed to monitor unauthorized genetically modified (GM) events in India. Most of the GM events included in this study are either authorized for commercial cultivation or field trials, which were indigenously developed or imported for research purposes. The developed system consists of a 96-well prespotted plate with lyophilized primers and probes, for simultaneous detection of 47 targets in duplicate, including 21 event-specific sequences, 5 construct regions, 15 for transgenic elements, and 6 taxon-specific targets for cotton, eggplant, maize, potato, rice, and soybean. Limit of detection (LOD) of assays ranged from 0.1 to 0.01% GM content for different targets. Applicability, robustness, and practical utility of the developed system were verified with stacked GM cotton event, powdered samples of proficiency testing and two unknown test samples. This user-friendly multitarget approach can be efficiently utilized for monitoring the unauthorized GM events in an Indian context.

Qualitative and Quantitative PCR-Based Detection Methods for Authorized Genetically Modified Cotton Events in India.

Qualitative diagnostics for all five commercialized genetically modified (GM) cotton events for insect resistance in India is being reported for the first time in this paper. The cost-effective and robust multiplex PCR (MPCR)-based detection assay, distinguishing the insect resistant transgenic Bt cotton events, viz., MON531, MON15985, Event 1, GFM-cry1A, and MLS-9124, has been developed. This decaplex PCR assay targets nine transgenic elements, viz., sequences of four transgenes, three transgene constructs, and two event-specific sequences along with one endogenous reference gene. The LOD of the qualitative MPCR assay was up to 0.1%. A quantitative detection method for four widely commercially cultivated GM cotton events, namely, MON531, MON15985, Event 1, and GFM-cry1A, covering 99.5% of the total area under GM cultivation in the country, is also reported. A construct-specific real-time PCR assay has been developed for quantification of these GM cotton events with LOQ <0.05% and LOD <0.025%. The developed assays will be of great use to screen for the presence/absence of authorized GM cotton events in unknown samples and to check the authenticity of GM cotton seed samples.

Visual and Real-Time Event-Specific Loop-Mediated Isothermal Amplification Based Detection Assays for Bt Cotton Events MON531 and MON15985.

Bt cotton events MON531 and MON15985 are authorized for commercial cultivation in more than 18 countries. In India, four Bt cotton events have been commercialized; more than 95% of total area under genetically modified (GM) cotton cultivation comprises events MON531 and MON15985. The present study reports on the development of efficient event-specific visual and real-time loop-mediated isothermal amplification (LAMP) assays for detection and identification of cotton events MON531 and MON15985. Efficiency of LAMP assays was compared with conventional and real-time PCR assays. Real-time LAMP assay was found time-efficient and most sensitive, detecting up to two target copies within 35 min. The developed real-time LAMP assays, when combined with efficient DNA extraction kit/protocol, may facilitate onsite GM detection to check authenticity of Bt cotton seeds.

DNA-Based Diagnostics for Genetically Modified Cotton: Decaplex PCR Assay to Differentiate MON531 and MON15985 Bt Cotton Events

The adoption rate and global area under cultivation of genetically modified (GM) crops is dramatically increasing in recent past. GM cotton has occupied 25.0 million hectares (mha) comprising 15.6% of the global area under GM cultivation. Bt cotton, expressing delta-endotoxins from Bacillus thuringiensis (Bt), is the only commercialized crop in India that is planted on an area of 10.6 mha. With the increase in development and commercialization of GM crops, it is necessary to develop appropriate qualitative and quantitative methods for detection of different GM events. Robust diagnostics for GM detection need to be developed and implemented to monitor and detect different events of GM cotton in India. This chapter summarizes the methods based on polymerase chain reaction (PCR) being employed for detection of different GM events of cotton. We describe a decaplex PCR method for identification and differentiation of two major commercialized events of Bt cotton, i.e., MON531 and MON15985, in India.

Qualitative and Quantitative Diagnostics for EE1 Event of Bt Eggplant

The genetically modified (GM) Bt eggplant event EE1 expresses Cry1Ac protein, conferring resistance against fruit and shoot borer. Although the field trials of this event under biosafety research levels I and II (BRL-I and II) have already been conducted, it has not yet been commercialized in India. In this chapter, various strategies for efficient screening, detection, and quantification of EE1 event are discussed in detail. The screening strategies involve polymerase chain reaction (PCR)/multiplex PCR, matrix approach, and loop-mediated isothermal amplification. The detection technologies are based on qualitative PCR, quantitative real-time PCR, and multitarget real-time PCR (where EE1 event can be simultaneously targeted with other major GM events of different crops). The reported strategies can be employed for detection and quantification of Bt eggplant and would be useful for monitoring the adventitious presence of transgenes and to ensure GM-free conservation of eggplant germplasm in genebanks.

Monitoring adventitious presence of transgenes in ex situ okra (Abelmoschus esculentus) collections conserved in genebank: a case study

The National Gene Bank at ICAR-National Bureau of Plant Genetic Resources, New Delhi, second largest genebank in the world, conserves more than 0.4 million accessions, including 3434 accessions of okra. Wild and weedy relatives of crops are being conserved as ex situ collections in the genebanks or on farm in the diversity-rich delicate pockets. To ensure GM-free conservation of the germplasm, unintentional introgression of transgenes needs to be monitored. In India, field trials of Bt okra with cry1Ac gene at Biosafety Research Levels-I and II were conducted by Maharashtra Hybrid Seeds Company Limited during 2006–2007. For preliminary study, adventitious presence of transgenes in 50 okra accessions of conserved ex situ collections was undertaken. A two step-approach was employed, first step comprising screening assays targeting Cauliflower Mosaic Virus 35S (CaMV35S) promoter and Agrobacterium tumefaciensnos terminator using singleplex Polymerase Chain Reaction (PCR) and real-time PCR assays; followed by the second step employing gene-specific assay targeting cry1Ac gene. Based on the present study, no adventitious presence of transgenes was detected in tested samples. The present study would be efficient and reliable approach for monitoring adventitious presence or unintentional introgression of transgenes in larger collections of okra and in collections of other crops, e.g., eggplant, rice, exhibiting rich diversity, where field trials of respective GM crops are being conducted in close proximity of their wild and weedy relatives.