Comparison of bacterial diversity, root exudates and soil enzymatic activities in the rhizosphere of AVP1-transgenic and nontransgenic wheat (Triticum aestivum L.).

AIMS: Soil microbial communities are among the most diverse communities that might be affected due to transgenic crops. Therefore, risk assessment studies on transgenes are essentially required as any adverse effects may depend not only on the specific gene and crop involved but also on soil conditions. METHODS AND RESULTS: The present study deals with the comparison of bacterial populations, root exudates and activities of soil enzymes in nontransgenic and AVP1-transgenic wheat rhizosphere, overexpressing vacuolar H + pyrophosphatase for salinity and drought stress tolerance. Amounts of organic acids and sugars produced as root exudates and activities of dehydrogenase, phosphatase and protease enzymes in soil solution showed no significant differences in AVP1-transgenic and nontransgenic wheat rhizosphere, except for urease and phenol oxidase activities. The higher copy number of nifH gene showed the abundance of nitrogen-fixing bacteria in the rhizosphere of AVP1-transgenic wheat compared with nontransgenic wheat. nifH gene sequence analysis indicated the common diazotrophic genera Azospirillum, Bradyrhizobium, Rhizobium and Pseudomonas in AVP1-transgenic and nontransgenic wheat except for Zoogloea detected only in nontransgenic wheat. Using 454-pyrosequencing of 16S rRNA gene from soil DNA, a total of 156, 282 sequences of 18 phyla were obtained, which represented bacterial (128,006), Archeal (7928) and unclassified (21,568) sequences. Proteobacteria, Crenarchaeota and Firmicutes were the most abundant phyla in the transgenic and nontransgenic wheat rhizosphere. Further comparison of different taxonomic units at the genus level showed similar distribution in transgenic and nontransgenic wheat rhizospheres. CONCLUSION: We conclude that the AVP1 gene in transgenic wheat has no apparent adverse effects on the soil environment and different bacterial communities. However, the bacterial community depends on several other factors, not only genetic composition of the host plants. SIGNIFICANCE OF THE STUDY: The present research supports introduction and cultivation of transgenic plants in agricultural systems without any adverse effects on indigenous bacterial communities and soil ecosystems.

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.

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.

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.

Engineering cotton (Gossypium hirsutum L.) for resistance to cotton leaf curl disease using viral truncated AC1 DNA sequences.

Several important biological processes are performed by distinct functional domains found on replication-associated protein (Rep) encoded by AC1 of geminiviruses. Two truncated forms of replicase (tAC1) gene, capable of expressing only the N-terminal 669 bp (5'AC1) and C-terminal 783 bp (3'AC1) nucleotides cloned under transcriptional control of the CaMV35S were introduced into cotton (Gossypium hirsutum L.) using LBA4404 strain of Agrobacterium tumefaciens to make use of an interference strategy for impairing cotton leaf curl virus (CLCuV) infection in transgenic cotton. Compared with nontransformed control, we observed that transgenic cotton plants overexpressing either N-terminal (5'AC1) or C-terminal (3'AC1) sequences confer resistance to CLCuV by inhibiting replication of viral genomic and ß satellite DNA components. Molecular analysis by Northern blot hybridization revealed high transgene expression in early and late growth stages associated with inhibition of CLCuV replication. Of the eight T(1) transgenic lines tested, six had delayed and minor symptoms as compared to nontransformed control lines which developed disease symptoms after 2-3 weeks of whitefly-mediated viral delivery. Virus biological assay and growth of T(2) plants proved that transgenic cotton plants overexpressing 5'- and 3'AC1 displayed high resistance level up to 72, 81%, respectively, as compared to non-transformed control plants following inoculation with viruliferous whiteflies giving significantly high cotton seed yield. Progeny analysis of these plants by polymerase chain reaction (PCR), Southern blotting and virus biological assay showed stable transgene, integration, inheritance and cotton leaf curl disease (CLCuD) resistance in two of the eight transgenic lines having single or two transgene insertions. Transgenic cotton expressing partial AC1 gene of CLCuV can be used as virus resistance source in cotton breeding programs aiming to improve virus resistance in cotton crop.

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.

Geminivirus AL2 and L2 proteins suppress transcriptional gene silencing and cause genome-wide reductions in cytosine methylation.

Geminiviruses replicate single-stranded DNA genomes through double-stranded intermediates that associate with cellular histone proteins. Unlike RNA viruses, they are subject to RNA-directed methylation pathways that target viral chromatin and likely lead to transcriptional gene silencing (TGS). Here we present evidence that the related geminivirus proteins AL2 and L2 are able to suppress this aspect of host defense. AL2 and L2 interact with and inactivate adenosine kinase (ADK), which is required for efficient production of S-adenosyl methionine, an essential methyltransferase cofactor. We demonstrate that the viral proteins can reverse TGS of a green fluorescent protein (GFP) transgene in Nicotiana benthamiana when overexpressed from a Potato virus X vector and that reversal of TGS by geminiviruses requires L2 function. We also show that AL2 and L2 cause ectopic expression of endogenous Arabidopsis thaliana loci silenced by methylation in a manner that correlates with ADK inhibition. However, at one exceptional locus, ADK inhibition was insufficient and TGS reversal required the transcriptional activation domain of AL2. Using restriction-sensitive PCR and bisulfite sequencing, we showed that AL2-mediated TGS suppression is accompanied by reduced cytosine methylation. Finally, using a methylation-sensitive single-nucleotide extension assay, we showed that transgenic expression of AL2 or L2 causes global reduction in cytosine methylation. Our results provide further evidence that viral chromatin methylation is an important host defense and allow us to propose that as a countermeasure, geminivirus proteins reverse TGS by nonspecifically inhibiting cellular transmethylation reactions. To our knowledge, this is the first report that viral proteins can inhibit TGS.

Pepper leaf curl Lahore virus requires the DNA B component of Tomato leaf curl New Delhi virus to cause leaf curl symptoms.

BACKGROUND: Begomoviruses are whitefly-transmitted geminiviruses with genomes that consist of either two components (known as DNA A and DNA B) or a single component (homologous to the DNA A component of bipartite begomoviruses). Monopartite begomoviruses are often associated with a symptom-modulating DNA satellite (collectively known as betasatellites). Both bipartite and monopartite begomoviruses with associated satellites have previously been identified in chillies showing leaf curl symptoms in Pakistan. RESULTS: A chilli plant (Capsicum annum) with chilli leaf curl disease symptoms was found to contain a begomovirus, a betasatellite and the DNA B component of Tomato leaf curl New Delhi virus (ToLCNDV). The begomovirus consisted of 2747 nucleotides and had the highest sequence identity (99%) with Pepper leaf curl Lahore virus (PepLCLV-[PK: Lah:04], acc. no. AM404179). Agrobacterium-mediated inoculation of the clone to Nicotiana benthamiana, induced very mild symptoms and low levels of viral DNA, detected in systemically infected leaves by PCR. No symptoms were induced in Nicotiana tabacum or chillies either in the presence or absence of a betasatellite. However, inoculation of PepLCLV with the DNA B component of ToLCNDV induced leaf curl symptoms in N. benthamiana, N. tabacum and chillies and viral DNA accumulated to higher levels in comparison to plants infected with just PepLCLV. CONCLUSIONS: Based on our previous efforts aimed at understanding of diversity of begomoviruses associated with chillies, we propose that PepLCLV was recently mobilized into chillies upon its interaction with DNA B of ToLCNDV. Interestingly, the putative rep-binding iterons found on PepLCLV (GGGGAC) differ at two base positions from those of ToLCNDV (GGTGTC). This is the first experimental demonstration of the infectivity for a bipartite begomovirus causing chilli leaf curl disease in chillies from Pakistan and suggests that component capture is contributing to the emerging complexity of begomovirus diseases in the region.

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.

Embryogenic Calli Explants and Silicon Carbide Whisker-Mediated Transformation of Cotton (Gossypium hirsutum L.).

Rapid growth in the genetic transformation of plants is the outcome of versatile transformation methods, explant nature, and media regimes. Modern biotechnologists have now a toolkit embraced with different plant transformation methods to generate specific and targeted genetic variation for performance improvement of crop plants. Genetic information are created by proper custom synthesis/amplification of DNA sequences from natural sources, modification during gene cloning, and choice of regulatory sequences and delivered to plants via different plant transformation techniques. Cotton is known by different names like king of fiber crops, white gold, etc., due to its socioeconomic involvement in society livelihood. So cotton is the host of several transgenes delivered for the purpose of trait development of improvement outcompeting its wild counterparts. At present most of the cotton adopted by farmers is biotech and contributes significantly in meeting farmers and industry demands. It is the versatile nature of cotton that it has been subjected to different genetic transformation methods to provide the breeders with an opportunity to develop alien traits or improve the endogenous gene performance that are very difficult or impossible to develop through conventional breeding methods. Landmark achievements were achieved by expanding explant choice such as calli as explants as it reduces the extent of labor, time, and effort and thereby becoming cost-effective cotton transformation. Cotton calli becomes differentiated into embryogenic and non-embryogenic which requires regular screening, keeping in view texture, color, and growth behavior. Here we describe the calli features which are peculiar in nature when used as explants in a novel physical way of cotton transformation with different genes by using embryogenic calli as continuous source of explants. The dawn of genome editing has opened another horizon in transformation research and development enhancing the scope of cotton transformation.

Risk assessment of genetically modified sugarcane expressing AVP1 gene.

Biosafety is a multidisciplinary approach that encompasses social, societal, ethical issues and policies for the regulations of genetically modified (GM) organisms. The potential health risks associated with GM sugarcane containing AVP1 gene confers resistance against drought and salinity were evaluated by animal feeding studies and some genotoxicity assays. Acute and sub-chronic toxicity examinations were carried out via oral dose administration of GM sugarcane juice supplemented with the normal diet (modified from certified rodent standard diet) on Wistar rats. AVP1 protein concentration in sugarcane juice was 1mg/1 mL. Biochemical, haematological blood analyses were performed and the results revealed that there were non-significant differences among all the treatment groups; GM sugarcane juice, non-GM sugarcane juice and the control group (normal diet and water). Genotoxicity assessment based on the comet assay and the micronucleus assay data exhibited that AVP1 GM sugarcane was not genotoxic or cytotoxic in rat's peripheral blood. These research findings supported the conclusion that GM AVP1 sugarcane was non-toxic in experimental animals. Therefore, data generated through this research work would be helpful for the commercial release of GM AVP1 sugarcane.

Development and evaluation of double gene transgenic cotton lines expressing Cry toxins for protection against chewing insect pests.

Cotton is the main fiber producing crop globally, with a significant impact on the economy of Pakistan. Bt cotton expressing a Cry1Ac gene is grown over a large area in Pakistan, however, there is a major concern that bollworms may develop resistance. Here we have used a durable resistance strategy against bollworms by developing a double gene construct containing Cry1Ac and Cry2Ab (pGA482-12R) for cotton transformation. Both Cry toxin genes have been cloned in the same T-DNA borders and transferred successfully into cotton via Agrobacterium-mediated transformation. Both genes are expressed in transgenic cotton plants and is likely to help breeders in developing new cotton cultivars by incorporating these genes in cotton lines having no Bt genes or expressing Cry1Ac gene (Mon 531). Positive transgenic cotton was identified by PCR using specific primers for the amplification of both Cry1Ac and Cry2Ab genes. Cry1Ac and Cry2Ab expression was confirmed with an immunostrip test and quantified using ELISA that showed significant spatio-temporal expression of Cry2Ab ranging from 3.28 to 7.72 µg/g of the tissue leaf. Insect bioassay with army worm (Spodoptera litura) was performed to check the efficacy of NIBGE (National Institute for Biotechnology and Genetic Engineering) double gene transgenic cotton plants and up to 93% insect mortality was observed.

Silicon carbide whisker-mediated embryogenic callus transformation of cotton (Gossypium hirsutum L.) and regeneration of salt tolerant plants.

A silicon carbide whisker-mediated gene transfer system with recovery of fertile and stable transformants was developed for cotton (Gossypium hirsutum L.) cv. Coker-312. Two-month-old hypocotyl-derived embryogenic/non-embryogenic calli at different days after subculture were treated with silicon carbide whiskers for 2 min in order to deliver pGreen0029 encoding GUS gene and pRG229 AVP1 gene, encoding Arabidopsis vacuolar pyrophosphatase, having neomycin phosphotransferaseII (nptII) genes as plant-selectable markers. Three crucial transformation parameters, i.e., callus type, days after subculture and selection marker concentration for transformation of cotton calli were evaluated for optimum efficiency of cotton embryogenic callus transformation giving upto 94% transformation efficiency. Within six weeks, emergence of kanamycin-resistant (kmr) callus colonies was noted on selection medium. GUS and Southern blot analysis showed expression of intact and multiple transgene copies in the transformed tissues. Kanamycin wiping of leaves from T1, T2, and T3 progeny plants revealed that transgenes were inherited in a Mendelian fashion. Salt treatment of T1 AVP1 transgenic cotton plants showed significant enhancement in salt tolerance as compared to control plants. Thus far, this is first viable physical procedure after particle bombardment available for cotton that successfully can be used to generate fertile cotton transformants.

RNAi-Mediated Simultaneous Resistance Against Three RNA Viruses in Potato.

RNA interference (RNAi) technology has been successfully applied in stacking resistance against viruses in numerous crop plants. During RNAi, the production of small interfering RNAs (siRNAs) from template double-standard RNA (dsRNA) derived from expression constructs provides an on-switch for triggering homology-based targeting of cognate viral transcripts, hence generating a pre-programmed immunity in transgenic plants prior to virus infection. In the current study, transgenic potato lines (Solanum tuberosum cv. Desiree) were generated, expressing fused viral coat protein coding sequences from Potato virus X (PVX), Potato virus Y (PVY), and Potato virus S (PVS) as a 600-bp inverted repeat expressed from a constitutive 35S promoter. The expression cassette (designated Ec1/p5941) was designed to generate dsRNAs having a hairpin loop configuration. The transgene insertions were confirmed by glufosinate resistance, gene-specific PCR, and Southern blotting. Regenerated lines were further assayed for resistance to virus inoculation for up to two consecutive crop seasons. Nearly 100% resistance against PVX, PVY, and PVS infection was observed in transgenic lines when compared with untransformed controls, which developed severe viral disease symptoms. These results establish the efficacy of RNAi using the coat protein gene as a potential target for the successful induction of stable antiviral immunity in potatoes.

Development of a Triple Gene Cry1Ac-Cry2Ab-EPSPS Construct and Its Expression in Nicotiana benthamiana for Insect Resistance and Herbicide Tolerance in Plants.

Insect pest complex, cotton leaf curl disease and weeds pose major threat to crop production worldwide, including Pakistan. To address these problems, in the present study a triple gene construct harboring Cry1Ac, Cry2Ab, and EPSPS cassettes has been developed for plant specifically in cotton transformation against lepidopteron insect-pests and weeds. Nicotiana benthamiana (tobacco) was used as a model system for characterization of this triple gene construct. The construct has been assembled in plant expression vector and transformed in N. benthamiana. In six transgenic tobacco lines the integration of Cry1Ac-Cry2Ab-EPSPS in tobacco genome was checked by PCR, while successful protein expression of all the three genes was confirmed through immunostrip assay. Efficacy of Cry1Ac and Cry2Ab was evaluated through insect bioassay using armyworm (Spodoptera littoralis). These transgenic tobacco plants showed significant insect mortality as compared to control plants during insect bioassay. Three out of six tested transgenic lines L3, L5, and L9 exhibited 100% mortality of armyworm, while three other lines L1, L10, and L7 showed 86, 80, and 40% mortality, respectively. This construct can readily be used with confidence to transform cotton and other crops for the development of insect resistant and herbicide tolerant transgenic plants. The transgenic crop plants developed using this triple gene construct will provide an excellent germplasm resource for the breeders to improve their efficiency in developing stable homozygous lines as all the three genes being in a single T-DNA border will inherit together.

Detection of Multiple Potato Viruses in the Field Suggests Synergistic Interactions among Potato Viruses in Pakistan.

Viral diseases have been a major limiting factor threating sustainable potato (Solanum tuberosum L.) production in Pakistan. Surveys were conducted to serologically quantify the incidence of RNA viruses infecting potato; Potato virus X (PVX), Potato virus Y (PVY), Potato virus S (PVS), Potato virus A (PVA), Potato virus M (PVM) and Potato leaf roll virus (PLRV) in two major potato cultivars (Desiree and Cardinal). The results suggest the prevalence of multiple viruses in all surveyed areas with PVY, PVS and PVX dominantly widespread with infection levels of up to 50% in some regions. Co-infections were detected with the highest incidence (15.5%) for PVX and PVS. Additionally the data showed a positive correlation between co-infecting viruses with significant increase in absorbance value (virus titre) for at least one of the virus in an infected plant and suggested a synergistic interaction. To test this hypothesis, glasshouse grown potato plants were challenged with multiple viruses and analyzed for systemic infections and symptomology studies. The results obtained conclude that multiple viral infections dramatically increase disease epidemics as compared to single infection and an effective resistance strategy in targeting multiple RNA viruses is required to save potato crop.

Silicon carbide whisker-mediated transformation of cotton (Gossypium hirsutum L.).

Plant transformation methods are invaluable biotechnological tools to generate specific and targeted genetic variation for performance improvement of crop plants. Genetic information is created by proper modification during gene cloning flanked by proper regulatory sequences and delivered to plants via -different plant transformation techniques. Due to being a multipurpose plant, cotton has been subjected to different genetic transformation methods to provide the breeders with an opportunity to develop alien traits or improve the endogenous gene performance that are very difficult or impossible to develop through conventional breeding methods. Here we describe the novel physical way of cotton transformation with different genes by using embryogeneic calli as continuous source of explants.