Assessment of Genomic Diversity and Selective Pressures in Crossbred Dairy Cattle of Pakistan.

Improving the low productivity levels of native cattle breeds in smallholder farming systems is a pressing concern in Pakistan. Crossbreeding high milk-yielding holstein friesian (HF) breed with the adaptability and heat tolerance of Sahiwal cattle has resulted in offspring that are well-suited to local conditions and exhibit improved milk yield. The exploration of how desirable traits in crossbred dairy cattle are selected has not yet been investigated. This study aims to provide the first overview of the selective pressures on the genome of crossbred dairy cattle in Pakistan. A total of eighty-one crossbred, thirty-two HF and twenty-four Sahiwal cattle were genotyped, and additional SNP genotype data for HF and Sahiwal were collected from a public database to equate the sample size in each group. Within-breed selection signatures in crossbreds were investigated using the integrated haplotype score. Crossbreds were also compared to each of their parental breeds to discover between-population signatures of selection using two approaches: cross-population extended haplotype homozygosity and fixation index. We identified several overlapping genes associated with production, immunity, and adaptation traits, including U6, TMEM41B, B4GALT7, 5S_rRNA, RBM27, POU4F3, NSD1, PRELID1, RGS14, SLC34A1, TMED9, B4GALT7, OR2AK3, OR2T16, OR2T60, OR2L3, and CTNNA1. Our results suggest that regions responsible for milk traits have generally experienced stronger selective pressure than others.

Genetic insights into crossbred dairy cattle of Pakistan: exploring allele frequency, linkage disequilibrium, and effective population size at a genome-wide scale.

Linkage disequilibrium (LD) affects genomic studies accuracy. High-density genotyping platforms identify SNPs across animal genomes, increasing LD evaluation resolution for accurate analysis. This study aimed to evaluate the decay and magnitude of LD in a cohort of 81 crossbred dairy cattle using the GGP_HDv3_C Bead Chip. After quality control, 116,710 Single Nucleotide Polymorphisms (SNPs) across 2520.241 Mb of autosomes were retained. LD extent was assessed between autosomal SNPs within a 10 Mb range using the r2 statistics. LD value declined as inter-marker distance increased. The average r2 value was 0.24 for SNP pairs < 10 kb apart, decreasing to 0.13 for 50-100 kb distances. Minor allele frequency (MAF) and sample size significantly impact LD. Lower MAF thresholds result in smaller r2 values, while higher thresholds show increased r2 values. Additionally, smaller sample sizes exhibit higher average r2 values, especially for larger physical distance intervals (> 50 kb) between SNP pairs. Effective population size and inbreeding coefficient were 150 and 0.028 for the present generation, indicating a decrease in genetic diversity over time. These findings imply that the utilization of high-density SNP panels and customized/breed-specific SNP panels represent a highly favorable approach for conducting genome-wide association studies (GWAS) and implementing genomic selection (GS) in the Bos indicus cattle breeds, whose genomes are still largely unexplored. Furthermore, it is imperative to devise a meticulous breeding strategy tailored to each herd, aiming to enhance desired traits while simultaneously preserving genetic diversity.

Deciphering host-pathogen interaction during Streptomyces spp. infestation of potato.

Potato crop, currently, is the staple food crop of about 1.3 billion global population. Potato is attaining even more admiration globally day by day owing to its public acceptability. However, potato sustainable production is distinctly challenged by multiple factors like diseases, pests and climate change etc. Among diseases, common scab is one of the prime threats to potato crop due to its soil-borne nature and versatility in phytotoxins' secretion. Common scab is caused multiple number of phytopathogenic streptomyces strains. Despite extensive research programs, researchers are still unable to identify a significant solution to this threat that is proliferating exceptional rate across the globe. To develop feasible remedies, adequate information regarding host-pathogen interaction should be available. This review possesses insights on existing pathogenic species, the evolution of novel pathogenic streptomyces spp. and phytotoxins produced by the pathogenic strains. Furthermore, which type of physiological, biochemical and genetic activities occur during pathogen's infestation of the host are also canvassed.

A critical look on CRISPR-based genome editing in plants.

Clustered regularly interspaced short palindromic repeats (CRISPR)-based genome editing, derived from prokaryotic immunity system, is rapidly emerging as an alternative platform for introducing targeted alterations in genomes. The CRISPR-based tools have been deployed for several other applications including gene expression studies, detection of mutation patterns in genomes, epigenetic regulation, chromatin imaging, etc. Unlike the traditional genetic engineering approaches, it is simple, cost-effective, and highly specific in inducing genetic variations. Despite its popularity, the technology has limitations such as off-targets, low mutagenesis efficiency, and its dependency on in-vitro regeneration protocols for the recovery of stable plant lines. Several other issues such as persisted CRISPR activity in subsequent generations, the potential for transferring to its wild type population, the risk of reversion of edited version to its original phenotype particularly in cross-pollinated plant species when released into the environment and the scarcity of validated targets have been overlooked. This article briefly highlights these undermined aspects, which may challenge the wider applications of this platform for improving crop genetics.

Expression patterns of cp4-epsps gene in diverse transgenic Saccharum officinarum L. genotypes.

Glyphosate, a functional analogue of phosphoenolpyruvate (PEP), blocks the shikimate pathway by inhibiting the enzyme 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS; EC 2.5.1.19) through interference with the conversion of (shikimate-3-phosphate) S3P and PEP to 5-enolpyruvylshikimate-3-phosphate (EPSP) and subsequently leads to plant death. This metabolic pathway possesses great potential to be used for development of herbicide resistant transgenic crops and here in this study, we wanted to check the expression potential of CP4-EPSPS gene in various sugarcane genotypes. A synthetic version of CP4-EPSPS gene synthesized commercially, cloned in pGreen0029 vector, was transformed into regenerable embryogenic calli of three different sugarcane cultivars HSF-240, S2003US-778 and S2003US-114 using biolistic gene transfer approach for comparative transcriptional studies. Transgenic lines screened by PCR analysis were subjected to Southern hybridization for checking transgene integration patterns. All the tested lines were found to contain multiple (3-6) insert copies. Putative transgenic plants produced the CP4-EPSPS protein which was detected using immunoblot analysis. The CP4-EPSPS transcript expression detected by qRT-PCR was found to vary from genotype to genotype and is being reported first time. In vitro glyphosate assay showed that transformed plants were conferring herbicide tolerance. It is concluded that different cultivars of sugarcane give variable expression of the same transgene and reasons for this phenomenon needs to be investigated.

Genetic manipulations in crops: Challenges and opportunities.

An alarming increase in the human population necessitates doubling the world food production in the next few decades. Although a number of possible biotechnological measures are under consideration, central to these efforts is the development of transgenic crops to produce more food, and the traits with which plants could better adapt to adverse environmental conditions in a changing climate. The emergence of new tools for the introduction of foreign genes into plants has increased both our knowledge and the capacity to develop transgenic plants. In addition, a better understanding of genetic modifications has allowed us to study the impact that genetically modified crop plants may have on the environment. This article discusses different techniques routinely used to carry out genetic modifications in plants while highlighting challenges with them, which future research must address to increase acceptance of GM crops for meeting food security challenges effectively.

Genetically transformed tobacco plants expressing synthetic EPSPS gene confer tolerance against glyphosate herbicide.

Glyphosate quashes the synthesis of 5-enolpyruvylshikimate-3- phosphate synthase (EPSPS) enzyme which intercedes the functioning of shikimate pathway for the production of aromatic amino acids. Herbicide resistant crops are developed using glyphosate insensitive EPSPS gene isolated from Agrobacterium sp. strain CP4, which give farmers a sustainable weed control option. Intentions behind this study were to design and characterize the synthetic herbicide resistant CP4-EPSPS gene in a model plant system and check the effectiveness of transformed tobacco against application of glyphosate. Putative transgenic plants were obtained from independent transformation events, and stable plant transformation, transgene expression and integration were demonstrated respectively by PCR, qRT-PCR and Southern hybridization. Gene transcript level and gene copy number (1-4) varied among the tested transgenic tobacco lines. Herbicide assays showed that transgenic plants were resistant to glyphosate after 12 days of spraying with glyphosate, and EPSPS activity remained at sufficient level to withstand the spray at 1000 ppm of the chemical. T1 plants analyzed through immunoblot strips and PCR showed that the gene was being translated into protein and transmitted to the next generation successfully. This codon optimized synthetic CP4-EPSPS gene is functionally equivalent to the gene for glyphosate resistance available in the commercial crops and hence we recommend this gene for transformation into commercial crops.

Transgenic cotton expressing Allium sativum leaf agglutinin exhibits resistance to whiteflies and aphids without negative effects on ladybugs.

Cotton (Gossypium hirsutum L.) is of great economic importance as a cultivated crop in many parts of the world. In addition to being a pillar of the textile industry, cotton and its byproducts are used for livestock feed, seed oil, and other products. Bacillus thuringiensis crystal toxin (Bt) expression in cotton provides effective protection against chewing insects but does not defend plants from piercing/sucking insect pests. With the aim to create transgenic plants with resistance against piercing/sucking pests, we used Agrobacterium-mediated genetic transformation of cotton cultivar Coker 312 to express the Allium sativum leaf agglutinin (ASLA) gene from the phloem-specific rolC promoter. The ASLA transgene was stably inherited and showed Mendelian segregation in the T1 generation. Transgenic lines, expressing the ASLA gene, showed explicit resistance against major sap-sucking pests. Green peach aphid (Myzus persicae Sulzer) choice assays showed that 75% of aphids preferred untransformed cotton plants relative to those expressing the ASLA gene. In detached leaf bioassays, plants expressing ASLA caused 82% aphid mortality and 44-53% reduction in fecundity. Clip cage bioassays with whiteflies (Bemisia tabaci Gennadius) showed 74-82% mortality and 44-60% decrease in fecundity due to ASLA gene expression. In whole plant bioassays, whiteflies showed 77% mortality and a 54% decrease in fecundity on ASLA transgenics. Importantly, we did not observe a negative effect of the ASLA gene on ladybugs (Coccinella septempunctata) that consumed these whiteflies. Together, our findings demonstrate the potential of ASLA-transgenic cotton for providing protection against two devastating insect pests, whiteflies and aphids. The ASLA-transgenic cotton appears promising for direct commercial cultivation besides serving as a potential genetic resource in recombination breeding.

Molecular characterization of a new synthetic cry2ab gene in Nicotiana tabacum.

A newly-synthesized cry2Ab gene was characterized in Nicotiana tabacum, before its further transformation in cotton. Synthetic cry2Ab gene was cloned in pGreen0029 and its expression was transiently analyzed at mRNA level through agroinfiltration in tobacco. The mRNA of cry2Ab was detected after 72 h agroinfiltration through PCR using total plant RNA. This construct was then transformed into N. tabacum through Agrobacterium. Insect bioassays were conducted on detached leaves using first instar Spodoptera exigua larvae; after 96 h significant insect mortality was recorded. This newly synthesized gene was effective in controlling S. exigua first instar larvae. It can be used in combinations with other Bt genes like cry1Ac for developing resistance against major insect pests of cotton and further widening the insect control spectrum.

Physiological responses induced by phospholipase C isoform 5 upon heat stress in Arabidopsis thaliana.

Plant's perception of heat stress involves several pathways and signaling molecules, such as phosphoinositide, which is derived from structural membrane lipids phosphatidylinositol. Phospholipase C (PLC) is a well-known signaling enzyme containing many isoforms in different organisms. In the present study, Phospholipase C Isoform 5 (PLC5) was investigated for its role in thermotolerance in Arabidopsis thaliana. Two over-expressing lines and one knock-down mutant of PLC5 were first treated at a moderate temperature (37 °C) and left for recovery. Then again exposed to a high temperature (45 °C) to check the seedling viability and chlorophyll contents. Root behavior and changes in 32Pi labeled phospholipids were investigated after their exposure to high temperatures. Over-expression of PLC5 (PLC5 OE) exhibited quick and better phenotypic recovery with bigger and greener leaves followed by chlorophyll contents as compared to wild-type (Col-0) and PLC5 knock-down mutant in which seedling recovery was compromised. PLC5 knock-down mutant illustrated well-developed root architecture under controlled conditions but stunted secondary roots under heat stress as compared to over-expressing PLC5 lines. Around 2.3-fold increase in phosphatidylinositol 4,5-bisphosphate level was observed in PLC5 OE lines upon heat stress compared to wild-type and PLC5 knock-down mutant lines. A significant increase in phosphatidylglycerol was also observed in PLC5 OE lines as compared to Col-0 and PLC5 knock-down mutant lines. The results of the present study demonstrated that PLC5 over-expression contributes to heat stress tolerance while maintaining its photosynthetic activity and is also observed to be associated with primary and secondary root growth in Arabidopsis thaliana.

Employing template-directed CRISPR-based editing of the OsALS gene to create herbicide tolerance in Basmati rice.

Rice (Oryza sativa) is one of the primary food crops which contributes major portion of daily calorie intake. It is used as model crop for various genome editing studies. Basmati rice was also explored for establishing non-homologous end joining-based genome editing. But it was not clear whether homology-directed repair (HDR)-based genome editing can be done in Basmati rice. The current study was designed to establish HDR-based genome editing in Basmati rice to develop herbicide tolerance. There is severe weed spread when rice is grown via direct planted rice method in various countries to save labour and water resources. Therefore, the use of herbicides is necessary to control weeds. These herbicides can also affect cultivated rice which creates the need to develop herbicide-tolerant rice. In current study, we introduced a point mutation in Acetolactate Synthase gene to convert tryptophan to leucine at position 548. For this purpose, different constructs for HDR were tested with different RNA scaffold and orientation of repair templates. Out of four different architectures, the one having repair template identical to the target DNA strand precisely edited the target site. We successfully established template-directed CRISPR-Cas9 system in Super Basmati rice by detecting desired substitutions at the target site in Acetolactate Synthase locus. Moreover, this editing of Acetolactate Synthase gene resulted in the production of herbicide tolerance in Super Basmati rice. This study suggests that such type of HDR system can be used to precisely edit other genes for crop improvement.

A source of resistance against yellow mosaic disease in soybeans correlates with a novel mutation in a resistance gene.

Yellow mosaic disease (YMD) is one of the major devastating constraints to soybean production in Pakistan. In the present study, we report the identification of resistant soybean germplasm and a novel mutation linked with disease susceptibility. Diverse soybean germplasm were screened to identify YMD-resistant lines under natural field conditions during 2016-2020. The severity of YMD was recorded based on symptoms and was grouped according to the disease rating scale, which ranges from 0 to 5, and named as highly resistant (HR), moderately resistant (MR), resistant (R), susceptible (S), moderately susceptible (MS), and highly susceptible (HS), respectively. A HR plant named "NBG-SG Soybean" was identified, which showed stable resistance for 5 years (2016-2020) at the experimental field of the National Institute for Biotechnology and Genetic Engineering (NIBGE), Faisalabad, Pakistan, a location that is a hot spot area for virus infection. HS soybean germplasm were also identified as NBG-47 (PI628963), NBG-117 (PI548655), SPS-C1 (PI553045), SPS-C9 (PI639187), and cv. NARC-2021. The YMD adversely affected the yield and a significant difference was found in the potential yield of NBG-SG-soybean (3.46 ± 0.13a t/ha) with HS soybean germplasm NARC-2021 (0.44 ± 0.01c t/ha) and NBG-117 (1.12 ± 0.01d t/ha), respectively. The YMD incidence was also measured each year (2016-2020) and data showed a significant difference in the percent disease incidence in the year 2016 and 2018 and a decrease after 2019 when resistant lines were planted. The resistance in NBG-SG soybean was further confirmed by testing for an already known mutation (SNP at 149th position) for YMD in the Glyma.18G025100 gene of soybean. The susceptible soybean germplasm in the field was found positive for the said mutation. Moreover, an ortholog of the CYR-1 viral resistance gene from black gram was identified in soybean as Glyma.13G194500, which has a novel deletion (28bp/90bp) in the 5`UTR of susceptible germplasm. The characterized soybean lines from this study will assist in starting soybean breeding programs for YMD resistance. This is the first study regarding screening and molecular analysis of soybean germplasm for YMD resistance.

Genome-Wide Identification and Analysis of the Hsp40/J-Protein Family Reveals Its Role in Soybean (Glycine max) Growth and Development.

The J-protein family comprises molecular chaperones involved in plant growth, development, and stress responses. Little is known about this gene family in soybean. Hence, we characterized J-protein genes in soybean, with the most highly expressed and responsive during flower and seed development. We also revealed their phylogeny, structure, motif analysis, chromosome location, and expression. Based on their evolutionary links, we divided the 111 potential soybean J-proteins into 12 main clades (I-XII). Gene-structure estimation revealed that each clade had an exon-intron structure resembling or comparable to others. Most soybean J-protein genes lacked introns in Clades I, III, and XII. Moreover, transcriptome data obtained from a publicly accessible soybean database and RT-qPCR were used to examine the differential expression of DnaJ genes in various soybean tissues and organs. The expression level of DnaJ genes indicated that, among 14 tissues, at least one tissue expressed the 91 soybean genes. The findings suggest that J-protein genes could be involved in the soybean growth period and offer a baseline for further functional research into J-proteins' role in soybean. One important application is the identification of J-proteins that are highly expressed and responsive during flower and seed development in soybean. These genes likely play crucial roles in these processes, and their identification can contribute to breeding programs to improve soybean yield and quality.

Development of broad-spectrum insect-resistant tobacco by expression of synthetic cry1Ac and cry2Ab genes.

Efficacy of two newly synthesized cry1Ac and cry2Ab genes was checked in tobacco before their expression in cotton. Both genes were artificially synthesized and codon optimized with respect to cotton-preferred codon usage. These genes were cloned in a plant expression vector and then transformed into tobacco. Fifty-eight putative transgenic plants were recovered from the selected explants. Successful integration of both genes in plant genome was confirmed by PCR amplification. Expression of transgenes was confirmed by PCR amplification from total plant RNA. Detached leaf insect bioassays were conducted with Helicoverpa armigera and Spodoptera exigua larvae. About 12 % of the transgenic plants showed significantly high resistance to S. exigua. Significant mortality (62 %) of H. armigera was recorded within 24 h of bioassays. Both toxins showed synergistic effect in tobacco and broadened the spectrum of plant activity against insects.

Development and evaluation of triple gene transgenic cotton lines expressing three genes (Cry1Ac-Cry2Ab-EPSPS) for lepidopteran insect pests and herbicide tolerance.

Cotton is an international agricultural commodity and the main cash crop of Pakistan of which quality and quantity are subject to various whims of nature. Climate change, insect pest complex, and weeds are reducing its productivity. Here, we have developed triple gene cotton containing EPSPS gene along with two Bt toxin genes Cry1Ac and Cry2Ab using a strategy where all three genes are cloned in the same T-DNA, followed by successful cotton transformation via Agrobacterium-mediated transformation. This strategy has been developed to help cotton breeders in developing new cultivars by incorporating these genes into the non-transgenic or single Bt (Cry1Ac) gene cotton background where all three genes will inherit together. The expression of all three proteins was confirmed through immunostrips and was quantified through enzyme-linked immunosorbent assay (ELISA). The spatio-temporal expression of Bt protein in different parts of triple gene NIBGE cotton plants was determined. Maximum expression was found in leaves followed by seeds and boll rinds. Insect bioassays with cotton bollworms (Helicoverpa armigera), armyworms (Spodoptera litura), and pink bollworms (Pectinophora gossypiella) showed more than 90% mortality. The best performing line (NIBGE-E2) on the basis of spatiotemporal expression, glyphosate assays, and insect mortality data, was used for event characterization by using the genome sequencing approach. The event was successfully characterized and named NIBGE 20-01. A diagnostics test based on event-specific PCR was developed and its ability to distinguish NIBGE 20-01 event from other commercial transgenic cotton events was confirmed. To confirm stable expression of all three proteins in the field conditions, homozygous transgenic lines were grown in the field and the expression was confirmed through immunostrip assays. It was found that all three genes are expressed under field conditions. To show that all three genes are inherited together upon crossing with local elite cotton lines, the F1 generation was grown under glasshouse and field conditions. The expression of all three genes was confirmed under field conditions. Our results showed that transgenic cotton with three genes cloned in the same T-DNA can express all genes and can be conveniently transferred into elite cotton lines through a single cross.

Smart breeding approaches in post-genomics era for developing climate-resilient food crops.

Improving the crop traits is highly required for the development of superior crop varieties to deal with climate change and the associated abiotic and biotic stress challenges. Climate change-driven global warming can trigger higher insect pest pressures and plant diseases thus affecting crop production sternly. The traits controlling genes for stress or disease tolerance are economically imperative in crop plants. In this scenario, the extensive exploration of available wild, resistant or susceptible germplasms and unraveling the genetic diversity remains vital for breeding programs. The dawn of next-generation sequencing technologies and omics approaches has accelerated plant breeding by providing the genome sequences and transcriptomes of several plants. The availability of decoded plant genomes offers an opportunity at a glance to identify candidate genes, quantitative trait loci (QTLs), molecular markers, and genome-wide association studies that can potentially aid in high throughput marker-assisted breeding. In recent years genomics is coupled with marker-assisted breeding to unravel the mechanisms to harness better better crop yield and quality. In this review, we discuss the aspects of marker-assisted breeding and recent perspectives of breeding approaches in the era of genomics, bioinformatics, high-tech phonemics, genome editing, and new plant breeding technologies for crop improvement. In nutshell, the smart breeding toolkit in the post-genomics era can steadily help in developing climate-smart future food crops.

Heterologous expression of a gene for thermostable xylanase from Chaetomium thermophilum in Pichia pastoris GS115.

We studied heterologous expression of xylanase 11A gene of Chaetomium thermophilum in Pichia pastoris and characterized the thermostable nature of the purified gene product. For this purpose, the xylanase 11A gene of C. thermophilum was cloned in P. pastoris GS115 under the control of AOX1 promoter. The maximum extracellular activity of recombinant xylanase (xyn698: gene with intron) was 15.6 U ml(-1) while that of recombinant without intron (xyn669) was 1.26 U ml(-1) after 96 h growth. The gene product was purified apparently to homogeneity level. The optimum temperature of pure recombinant xylanase activity was 70°C and the enzyme retained its 40.57% activity after incubation at 80°C for 10 min. It exhibited quite lower demand of activation energy, enthalpy, Gibbs free energy, entropy, and xylan binding energy during substrate hydrolysis than that required by that of the donor, thus indicating its thermostable nature. pH-dependent catalysis showed that it was quite stable in a pH range of 5.5-8.5. This revealed that gene was successfully processed in P. pastoris and remained heat stable and may qualify for its potential use in paper and pulp and animal feed applications.

Spider toxin (Hvt) gene cloned under phloem specific RSs1 and RolC promoters provides resistance against American bollworm (Heliothis armigera).

Spider venoms are neurotoxin proteins that can kill insects. Spider toxin Hvt gene was cloned under two phloem specific RSs1 and RolC promoters, transformed into tobacco plants through Agrobacterium-mediated transformation and tested against Heliothis armigera larvae. Transgenic plants were confirmed through PCR. First instar larvae of H. armigera were released on detached leaves of transformed and non-transformed plants. Insect bioassays showed 93-100% mortality of H. armigera larvae within 72 h on the leaves of transgenic plants while all larvae survived and continued feeding on detached leaves from non-transformed control plants. The Hvt gene expressing under phloem specific RSs1 and RolC promoters could therefore be used for developing H. armigera-resistant, genetically-modified crops.

Development of event-specific detection method for identification of insect resistant NIBGE-1601 cotton harboring double gene Cry1Ac-Cry2Ab construct.

Bt cotton expressing Cry1Ac is being cultivated in Pakistan. It has been observed that pink bollworm may have developed resistance against single Bt gene (Cry1Ac). For durable resistance, insect resistant NIBGE-1601 cotton harboring double gene Cry1Ac-Cry2Ab construct was developed. There was a need to characterize NIBGE-1601 event for intellectual property rights protection. The Presence of NIBGE Cry1Ac and NIBGE Cry2Ab genes was checked in NIBGE-1601 cotton plants through PCR, while there was no amplification using primers specific for Monsanto events (MON531, MON15985, MON1445). Using genome walking technology, NIBGE-601 event has been characterized. Event-specific primers of NIBGE-1601 were designed and evaluated to differentiate it from other cotton events mentioned above. NIBGE-1601 event detection primers are highly specific, therefore, can detect NIBGE 1601 event at different conditions using single or multiplex PCR. In the qualitative PCR, using NIBGE-1601 event specific primers, 0.05 ng was the limit of detection for NIBGE-1601double gene cotton genomic DNA. Thus event characterization and development of event-specific diagnostics will help in breeding new cotton varieties resistant to cotton bollworms.

Comparison of in Vitro and in Planta Toxicity of Vip3A for Lepidopteran Herbivores.

Agricultural pest infestation is as old as domestication of food crops and contributes a major share to the cost of crop production. In a transgenic pest control approach, plant production of Vip3A, an insecticidal protein from Bacillus thuringiensis, is effective against lepidopteran pests. A synthetic Vip3A gene was evaluated for efficacy against Spodoptera litura Fabricius (Lepidoptera: Noctuidae; cotton leafworm), Spodoptera exigua Hübner (Lepidoptera: Noctuidae; beet armyworm), Spodoptera frugiperda Smith (Lepidoptera: Noctuidae; fall armyworm), Helicoverpa armigera Hübner (Lepidoptera: Noctuidae; cotton bollworm), Helicoverpa zea Boddie (Lepidoptera: Noctuidae; corn earworm), Heliothis virescens Fabricius (Lepidoptera: Noctuidae; tobacco budworm), and Manduca sexta L. (Lepidoptera: Sphingidae; tobacco hornworm) in tobacco. In artificial diet assays, the concentration required to achieve 50% mortality was highest for H. zea followed by H. virescens > S. exigua > H. armigera > M. sexta > S. frugiperda > S. litura. By contrast, in bioassays with detached leaves from Vip3A transgenic tobacco, the time until 50% lethality was M. sexta > H. virescens > S. litura > H. zea > H. armigera > S. exigua. There was no significant correlation between the artificial diet and transgenic plant bioassay results. Notably, the two insect species that are best-adapted for growth on tobacco, M. sexta and H. virescens, showed the greatest time to 50% mortality on Vip3A-transgenic tobacco. Together, our results suggest that artificial diet assays may be a poor predictor of Vip3A efficacy in transgenic plants, lepidopteran species vary in their sensitivity to Vip3A in diet-dependent manner, and host plant adaptation of the targeted herbivores should be considered when designing transgenic plants for pest control.