Multiplex Cas9-based excision of CLCuV betasatellite and DNA-A revealed reduction of viral load with asymptomatic cotton plants.

MAIN CONCLUSION: Multiplexed Cas9-based genome editing of cotton resulted in reduction of viral load with asymptomatic cotton plants. In depth imaging of proteomic dynamics of resulting CLCuV betasatellite and DNA-A protein was also performed. The notorious  cotton leaf curl virus (CLCuV), which is transmitted by the sap-sucking insect whitefly, continuously damages cotton crops. Although the application of various toxins and RNAi has shown some promise, sustained control has not been achieved. Consequently, CRISPR_Cas9 was applied by designing multiplex targets against DNA-A (AC2 and AC3) and betasatellite (ßC1) of CLCuV using CRISPR direct and ligating into the destination vector of the plant using gateway ligation method. The successful ligation of targets into the destination vector was confirmed by the amplification of 1049 bp using a primer created from the promoter and target, while restriction digestion using the AflII and Asc1 enzymes determined how compact the plasmid developed and the nucleotide specificity of the plasmid was achieved through Sanger sequencing. PCR confirmed the successful introduction of plasmid into CKC-1 cotton variety. Through Sanger sequencing and correlation with the mRNA expression of DNA-A and betasatellite in genome-edited cotton plants subjected to agroinfiltration of CLCuV infectious clone, the effectiveness of knockout was established. The genome-edited cotton plants demonstrated edited efficacy of 72% for AC2 and AC3 and 90% for the (ßC1) through amplicon sequencing, Molecular dynamics (MD) simulations were used to further validate the results. Higher RMSD values for the edited ßC1 and AC3 proteins indicated functional loss caused by denaturation. Thus, CRISPR_Cas9 constructs can be rationally designed using high-throughput MD simulation technique. The confidence in using this technology to control plant virus and its vector was determined by the knockout efficiency and the virus inoculation assay.

CRISPR/Cas-mediated knockdown of vacuolar invertase gene expression lowers the cold-induced sweetening in potatoes.

MAIN CONCLUSION: VInv gene editing in potato using CRISPR/Cas9 resulted in knockdown of expression and a lower VInv enzymatic activity resulting in a decrease in post-harvest cold-storage sugars formation and sweetening in potatoes. CRISPR-Cas9-mediated knockdown of vacuolar invertase (VInv) gene was carried out using two sgRNAs in local cultivar of potato plants. The transformation efficiency of potatoes was found to be 11.7%. The primary transformants were screened through PCR, Sanger sequencing, digital PCR, and ELISA. The overall editing efficacy was determined to be 25.6% as per TIDE analysis. The amplicon sequencing data showed maximum indel frequency for potato plant T12 (14.3%) resulting in 6.2% gene knockout and 6% frame shift. While for plant B4, the maximum indel frequency of 2.0% was found which resulted in 4.4% knockout and 4% frameshift as analyzed by Geneious. The qRT-PCR data revealed that mRNA expression of VInv gene was reduced 90-99-fold in edited potato plants when compared to the non-edited control potato plant. Following cold storage, chips analysis of potatoes proved B4 and T12 as best lines. Reducing sugars' analysis by titration method determined fivefold reduction in percentage of reducing sugars in tubers of B4 transgenic lines as compared to the control. Physiologically genome-edited potatoes behaved like their conventional counterpart. This is first successful report of knockdown of potato VInv gene in Pakistan that addressed cold-induced sweetening resulting in minimum accumulation of reducing sugars in genome edited tubers.

Enhancing the resilience of transgenic cotton for insect resistance.

BACKGROUND: The efficacy of Bt crystal proteins has been compromised due to their extensive utilization in the field. The second-generation Bt vegetative insecticidal proteins could be the best-suited alternative to combat resistance build-up due to their broad range affinity with midgut receptors of insects. MATERIAL AND RESULTS: The codon-optimized synthetic vegetative insecticidal proteins (Vip3Aa) gene under the control of CaMV35S promoter was transformed into a locally developed transgenic cotton variety (CKC-01) expressing cry1Ac and cry2A genes. Transformation efficiency of 1.63% was recorded. The highest Vip3Aa expression (51.98-fold) was found in MS3 transgenic cotton plant. Maximum Vip3Aa protein concentration (4.23 µg/mL) was calculated in transgenic cotton plant MS3 through ELISA. The transgenic cotton plant (MS3) showed one copy number on both chromatids in the homozygous form at chromosome 8 at the telophase stage. Almost 99% mortality of H. armigera was recorded in transgenic cotton plants expressing double crystal proteins pyramided with Vip3Aa gene as contrasted to transgenic cotton plant expressing only double crystal protein with 70% mortality. CONCLUSIONS: The results obtained during this study suggest that the combination of Bt cry1Ac, cry2A, and Vip3Aa toxins is the best possible alternative approach to combat chewing insects.

Heterologous expression of cry1Ia12 insecticidal gene in cotton encodes resistance against pink bollworm, Pectinophora gossypiella (Lepidoptera: Gelechiidae); an alternate insecticidal gene for insect pest management.

BACKGROUND: Cotton is continuously exposed to sucking and chewing insect pest pressure since emergence to harvesting. Pink bollworm (Pectinophora gossypiella) has become major chewing insect pest to reduce the cotton yield and results in bad lint quality even in transgenic crops. The efficiency of insecticidal genes has been compromised due to extensive utilization of transgenic crops. METHODS AND RESULTS: The present study was conducted to evaluate the efficacy of an alternate cry1Ia12 insecticidal gene against pink bollworm (PBW) in cotton. Agrobacterium tumefaciens strain LBA4404 harboring pCAMBIA2300 expression vector containing cry1Ia12 gene under the control of 35S CaMV was used to transform a local cotton cultivar GS-01. The various molecular analyses revealed the transgene integration and expression in primary transformants. Among five selected transgenic plants, tcL-08 showed maximum (16.06-fold) mRNA expression of cry1Ia12 gene whereas tcL-03 showed minimum (2.33-fold) expression. Feeding bioassays of 2nd and 3rd instar pink bollworm (PBW) larvae on immature cotton bolls, flowers and cotton squares revealed up to 33.33% mortality on tcL-08 while lowest mortality (13.33%) was observed in tcL-03 and tcL-15. Furthermore, the average weight and size of survived larvae fed on transgenic plants was significantly lesser than the average weight of larvae survived on non-transgenic plants. CONCLUSIONS: The present study suggests the cry1Ia12 gene as an alternate insecticidal gene for the resistance management of cotton bollworms, especially PBW.

Enhanced expression of plasma membrane intrinsic protein 2 improves cotton fiber length in Gossypium arboreum.

BACKGROUND: Gossypium arboreum is a cotton crop native to tropical and subtropical regions that are naturally resistant to cotton leaf curl virus (CLCuV). However, its cultivation is unfavorable due to the lower quality and shorter fiber length of cotton when compared to the market leading G. hirsutum. Plasma membrane intrinsic protein 2 (PIP2) is an aquaporin responsible for the transport of water and small molecules across cellular membranes. This fluid transport influences cell elongation and cotton fibre development. Hence, increased PIP2 expression may yield plants with enhanced fiber qualities including length. METHODS AND RESULTS: To test this hypothesis, G. arboreum was transformed with a PIP2 gene construct (35SCpPIP2) using the Agrobacterium-mediated shoot apex cutting method. Relative expression of the CpPIP2 gene in transgenic plants increased up to 35-fold when compared with non-transgenic controls. Transgenic plants displayed a corresponding increase of staple length (up to 150%) when compared with non-transgenic controls. Transgene integration was examined using FISH and karyotyping and revealed the presence of a single transgene located on chromosome 6. CONCLUSION: Since G. arboreum is naturally whitefly and CLCuV resistant, this improvement of fiber length evidenced for CpPIP2 transgenic plants renders their crop production more economically viable.

Development of broad-spectrum and sustainable resistance in cotton against major insects through the combination of Bt and plant lectin genes.

KEY MESSAGE: Second generation Bt insecticidal toxin in comibination with Allium sativum leaf agglutinin gene has been successfully expressed in cotton to develop sustainable resistance against major chewing and sucking insects. The first evidence of using the Second-generation Bt gene in combination with Allium sativum plant lectin to develop sustainable resistance against chewing and sucking insects has been successfully addressed in the current study. Excessive use of Bt δ-endotoxins in the field is delimiting its insecticidal potential. Second-generation Bt Vip3Aa could be the possible alternative because it does not share midgut receptor sites with any known cry proteins. Insecticidal potential of plant lectins against whitefly remains to be evaluated. In this study, codon-optimized synthetic Bt Vip3Aa gene under CaMV35S promoter and Allium sativum leaf agglutinin gene under phloem-specific promoter were transformed in a local cotton variety. Initial screening of putative transgenic cotton plants was done through amplification, histochemical staining and immunostrip assay. The mRNA expression of Vip3Aa gene was increased to be ninefold in transgenic cotton line L6P3 than non-transgenic control while ASAL expression was found to be fivefold higher in transgenic line L34P2 as compared to non-transgenic control. The maximum Vip3Aa concentration was observed in transgenic line L6P3. Two copy numbers in homozygous form at chromosome number 9 and one copy number in hemizygous form at chromosome number 10 was observed in transgenic line L6P3 through fluorescent in situ hybridization. Significant variation was observed in transgenic cotton lines for morphological characteristics, whereas physiological parameters of plants and fiber characteristics (as assessed by scanning electron microscopic) remained comparable in transgenic and non-transgenic cotton lines. Leaf-detach bioassay showed that all the transgenic lines were significantly resistant to Helicoverpa armigera showing mortality rates between 78% and 100%. Similarly, up to 95% mortality of whiteflies was observed in transgenic cotton lines when compared with non-transgenic control lines.

Evaluation the effect of subchronic feeding of transgenic cotton line (CKC1) on the faecal microbiota of albino rabbits.

Recent studies have demonstrated a strong relationship between the intestinal microbiota and the host health. As such, consumers are increasingly becoming more concerned about the potential effect of certain foods/feeds, particularly of transgenic origin on the gut microbiota. Although the European Food Safety Authority has recommended in their guidelines, to study the effect of transgenic food/feed on host-microbiota, yet, few studies have focused on the evaluation of such effects mainly due to culturing difficulties. Therefore, this study was intended to evaluate the potential adverse effects of transgenic diet consumption on some specific gut microflora (Lactobacillus group, Bifidobacterium genus, Escherichia coli subgroup and Enterococcus genus) of rabbits. A total of forty-eight rabbits were randomly assigned into four groups and fed a diet containing a variable proportion of transgenic cottonseeds at 0, 20, 30 and 40% inclusion level, respectively. Changes in the specific or total faecal bacterial population were monitored at five different experimental stages (i.e. 0, 45, 90, 135 and 180 days) using both the traditional plate count method (TM) and quantitative real-time PCR (qPCR). No significant differences (p > .05) were observed concerning numbers of specific bacteria or total bacteria between the control and experimental groups, though qPCR showed numerically higher values in terms of 16S rRNA gene copies as compared to the values obtained from TM. However, such numerical differences were biologically insignificant (p > .05). Similarly, no significant variations were noticed in the calculated B/E (log10 copies of Bifidobacterium per g faces/log10 copies of E. coli genome per g faeces) ratios in all the groups. All the ratios were in the range of 1.24 to 1.30 throughout the experiment, indicating a good balance of intestinal microflora and greater resistance to intestinal disorders. It is therefore concluded that feeding transgenic cottonseeds could not adversely affect the gut microflora of rabbits during a long-term study.

Assessing the fate of recombinant plant DNA in rabbit's tissues fed genetically modified cotton.

Various feeding studies have been conducted with the different species of animals to evaluate the possible transfer of transgenic DNA (tDNA) from genetically modified (GM) feed into the animal tissues. However, the conclusions drawn from most of such studies are sometimes controversial. Thus, in the present study, an attempt has been made to evaluate the fate of tDNA in rabbits raised on GM cotton-based diet through PCR analysis of the DNA extracted specifically from blood, liver, kidney, heart and intestine (jejunum). A total of 48 rabbits were fed a mixed diet consisting variable proportions of transgenic cottonseeds meal (i.e. 0% w/w, 20% w/w, 30% w/w and 40% w/w) for 180 days. The presence of transgenic DNA fragments (Cry1Ac, Cry2A and CP4 EPSPS) or plant endogenous gene (Sad1) was traced in those specific tissues and organs. The presence of ß-actin (ACTB) was also monitored as an internal control. Neither the transgenic fragments (459 bp of Cry1Ac gene, 167 bp of Cry2A gene and111 bp of CP4 EPSPS gene) nor cotton endogenous reference gene (155 bp of Sad1) could be detected in any of the DNA samples extracted from the rabbit's tissues in both control and transgenic groups. However, 155 bp fragment of the rabbit's reference gene (ACTB) was recovered in all the DNA samples extracted from rabbit tissues. The results obtained from this study revealed that both plant endogenous and transgenic DNA fragments have same fate in rabbit's tissues and were efficiently degraded in the gastrointestinal tract (GIT).

Effect of dietary supplementation of recombinant Cry and Cp4 epsps proteins on haematological indices of growing rabbits.

Genetically modified (GM) crops expressing insect resistance and herbicide tolerance provide a novel approach for improved crop production but their advent at the same time presents serious challenges in terms of food safety. Although prevailing scientific proof has suggested that transgenic crops are analogous to their conventional counterparts, their use in human and animal diet gave rise to emotional public discussion. A number of studies had been conducted to evaluate the potential unintended effects of transgenic crops expressing single transgene, but very few studies for those with multiple transgenes. As the crops with single and multiple transgenes could impart different effects on non-target organisms, thus, risk evaluation of transgenic crops expressing more than one transgene is required to declare their biosafety. The present study was therefore designed to assess the effects of different levels of dietary transgenic cottonseed expressing recombinants proteins produced by Cry1Ac, Cry2A and Cp4epsps genes on haematological indices of growing rabbits. A total of 48 rabbits were assigned to four dietary treatments containing different levels of transgenic cottonseeds (i.e., 0% w/w, 20% w/w, 30% w/w and 40% w/w) with 0% w/w serving as control. Haematological parameters were measured at periodic intervals (0, 45, 90, 135 and 180) days. No significant (p > 0.05) dose-dependent effects were observed in most of the haematological parameters evaluated. Though, significant differences (p < 0.05) were recorded in the level of MCHC, MCH and HCT in some of experimental male and female rabbits, yet, they were not biologically significant, as all the differences were within the normal reference values. Our study suggested that feeding transgenic cottonseed of up to 40% could not adversely affect rabbit's haematological profile. However, further study needs to be conducted with different cotton genotypes expressing both single and polygenic traits before recommending the utilization of transgenic cottonseed in routine livestock feeding.

Designing and screening of universal drug from neem (Azadirachta indica) and standard drug chemicals against influenza virus nucleoprotein.

BACKGROUND: Different strains of influenza virus are affecting a large number of people worldwide. Many synthetic antiviral medicines are available for influenza virus in the market. But still there is a need for the development of universal drugs against these strains of influenza virus. METHODS: For this purpose conserved residues within the influenza virus nucleoprotein have been retrieved. The drugs, previously known to have antiviral properties, were screened to identify the best candidate universal drug against Influenza virus strains. Compounds from leaf extracts of neem, were also screened to identify the natural drugs without side effects. RESULT: Molecular docking identified three potential compounds (Nimbaflavone, Rutin, and Hyperoside) having perfect binding with reported conserved residues (ASP302, SER50) of influenza virus nucleoprotein that is involved in the binding of drugs. Further analysis showed Hyperoside as a universal drug against various influenza strains. Some chemical drugs were also evaluated through screening against nucleoprotein. The results showed six drugs (OMS, CBX, LGH, Naproxen, BMS-883559, and BMS-885838) which were interacting with same conserved residues (ASP302, TYR52, SER50, GLY288, SER376, and ARG99) as were found in the case of neem phytochemicals. Hyperoside from neem leaf extract along with drugs LGH, Naproxen, BMS-885838, and BMS-883559 showed best interactions with conserved residues of nucleoprotein. CONCLUSION: The compound Hyperoside from neem leaf extract along with drugs LGH, Naproxen, BMS-885838, and BMS-883559 showed best interactions with conserved residues of nucleoprotein. So these compounds have been identified for their potential against influenza strains to be utilized as a universal drug.

Risk assessment of Bt crops on the non-target plant-associated insects and soil organisms.

Transgenic plants containing Bacillus thuringiensis (Bt) genes are being cultivated worldwide to express toxic insecticidal proteins. However, the commercial utilisation of Bt crops greatly highlights biosafety issues worldwide. Therefore, assessing the risks caused by genetically modified crops prior to their commercial cultivation is a critical issue to be addressed. In agricultural biotechnology, the goal of safety assessment is not just to identify the safety of a genetically modified (GM) plant, rather to demonstrate its impact on the ecosystem. Various experimental studies have been made worldwide during the last 20 years to investigate the risks and fears associated with non-target organisms (NTOs). The NTOs include beneficial insects, natural pest controllers, rhizobacteria, growth promoting microbes, pollinators, soil dwellers, aquatic and terrestrial vertebrates, mammals and humans. To highlight all the possible risks associated with different GM events, information has been gathered from a total of 76 articles, regarding non-target plant and soil inhabiting organisms, and summarised in the form of the current review article. No significant harmful impact has been reported in any case study related to approved GM events, although critical risk assessments are still needed before commercialisation of these crops. © 2016 Society of Chemical Industry.

Chloroplast localization of Cry1Ac and Cry2A protein--an alternative way of insect control in cotton.

BACKGROUND: Insects have developed resistance against Bt-transgenic plants. A multi-barrier defense system to weaken their resistance development is now necessary. One such approach is to use fusion protein genes to increase resistance in plants by introducing more Bt genes in combination. The locating the target protein at the point of insect attack will be more effective. It will not mean that the non-green parts of the plants are free of toxic proteins, but it will inflict more damage on the insects because they are at maximum activity in the green parts of plants. RESULTS: Successful cloning was achieved by the amplification of Cry2A, Cry1Ac, and a transit peptide. The appropriate polymerase chain reaction amplification and digested products confirmed that Cry1Ac and Cry2A were successfully cloned in the correct orientation. The appearance of a blue color in sections of infiltrated leaves after 72 hours confirmed the successful expression of the construct in the plant expression system. The overall transformation efficiency was calculated to be 0.7%. The amplification of Cry1Ac-Cry2A and Tp2 showed the successful integration of target genes into the genome of cotton plants. A maximum of 0.673 µg/g tissue of Cry1Ac and 0.568 µg/g tissue of Cry2A was observed in transgenic plants. We obtained 100% mortality in the target insect after 72 hours of feeding the 2nd instar larvae with transgenic plants. The appearance of a yellow color in transgenic cross sections, while absent in the control, through phase contrast microscopy indicated chloroplast localization of the target protein. CONCLUSION: Locating the target protein at the point of insect attack increases insect mortality when compared with that of other transgenic plants. The results of this study will also be of great value from a biosafety point of view.

Biosafety and toxicity assessment of transgenic cotton-harboring insecticide and herbicide tolerant genes on albino mice.

Introduction: Genetic engineering has revolutionized agriculture by transforming biotic and abiotic stress-resistance genes in plants. The biosafety of GM crops is a major concern for consumers and regulatory authorities. Methodology: A 14-week biosafety and toxicity analysis of transgenic cotton, containing 5 transgenes ((Cry1Ac, Cry2A, CP4 EPSPS, VIP3Aa, and ASAL)), was conducted on albino mice. Thirty mice were divided into three groups (Conventional, Non-transgenic, without Bt, and transgenic, containing targeted crop) according to the feed given, with 10 mice in each group, with 5 male and 5 female mice in each group. Results: During the study, no biologically significant changes were observed in the non-transgenic and transgenic groups compared to the control group in any of the study's parameters i.e. increase in weight of mice, physiological, pathological, and molecular analysis, irrespective of the gender of the mice. However, a statistically significant change was observed in the hematological parameters of the male mice, while no such change was observed in the female study group mice. The expression analysis, however, of the TNF gene increases many folds in the transgenic group as compared to the non-transgenic and conventional groups. Conclusion: Overall, no physiological, pathological, or molecular toxicity was observed in the mice fed with transgenic feed. Therefore, it can be speculated that the targeted transgenic crop is biologically safe. However, more study is required to confirm the biosafety of the product on the animal by expression profiling.

Over-expression of GhACTIN1 under the control of GhSCFP promoter improves cotton fiber and yield.

Actin dynamics is pivotal in controlling cotton fiber elongation and the onset of secondary wall biosynthesis. We report that overexpression of GhACTIN1 under fiber fiber-specific promoter, GhSCFP, improves cotton fiber length, strength, and micronaire value. However, the effect of transgene has a more positive effect on fiber strength and micronaire value than fiber length. F-actin quantification and cellulose contents measurement in transgenic developing cotton fiber during the elongation phase showed an increase of up to 8.7% and 4.7% respectively. Additionally, physiological factors such as water use efficiency showed no significant change in transgenic cotton lines, while stomatal conductance and photosynthetic rate were significantly increased. Moreover, agronomical data determined that lint percentage (GOT) and seed cotton yield also increased up to 4.6% and 29.5% respectively, in transgenic cotton lines compared to the control lines. Our data demonstrate that the GhACTIN1 gene is a strong candidate gene for cotton fiber and yield improvement.

A sub-chronic feeding study of dual toxin insect-resistant transgenic maize (CEMB-413) on Wistar rats.

Genetically modified (GM) crops expressing insecticidal crystal proteins are widely accepted worldwide, but their commercial utilization demands comprehensive risk assessment studies. A 90-day risk assessment study was conducted on Wistar rats fed with GM maize (CEMB-413) expressing binary insect-resistant genes (cry1Ac and cry2Ab) at low (30%) and high (50%) dose along with a control diet group. The study used fifty Wistar rats randomly distributed in five treatment groups. Our study revealed that compared to controls, GM diet had no adverse effects on animal's health, including body weight, food consumption, clinical pathological parameters, serum hormone levels and histological parameters of testes and ovaries of rats. Differences were observed in transcripts levels of fertility related genes, but these were independent of treatment with GM diet.

Tissue specific expression of bacterial cellulose synthase (Bcs) genes improves cotton fiber length and strength.

Fiber growing inside the cotton bolls is a highly demandable product and its quality is key to the success of the textile industry. Despite the various efforts to improve cotton fiber staple length Pakistan has to import millions of bales to sustain its industrial needs. To improve cotton fiber quality Bacterial cellulose synthase (Bcs) genes (acsA, acsB) were expressed in a local cotton variety CEMB-00. In silico studies revealed a number of conserved domains both in the cotton-derived and bacterial cellulose synthases which are essential for the cellulose synthesis. Transformation efficiency of 1.27% was achieved by using Agrobacterium shoot apex cut method of transformation. The quantitative mRNA expression analysis of the Bcs genes in transgenic cotton fiber was found to be many folds higher during secondary cell wall synthesis stage (35 DPA) than the expression during elongation phase (10 DPA). Average fiber length of the transgenic cotton plant lines S-00-07, S-00-11, S-00-16 and S-00-23 was calculated to be 13.02% higher than that of the non-transgenic control plants. Likewise, the average fiber strength was found to be 20.92% higher with an enhanced cellulose content of 22.45%. The mutated indigenous cellulose synthase genes of cotton generated through application of CRISPR/Cas9 resulted in 6.03% and 12.10% decrease in fiber length and strength respectively. Furthermore, mature cotton fibers of transgenic cotton plants were found to have increased number of twists with smooth surface as compared to non-transgenic control when analyzed under scanning electron microscope. XRD analysis of cotton fibers revealed less cellulose crystallinity index in transgenic cotton fibers as compared to control fibers due to deposition of more amorphous cellulose in transgenic fibers as a result of Bcs gene expression. This study paved the way towards unraveling the fact that Bcs genes influence cellulose synthase activity and this enzyme helps in determining the fate of cotton fiber length and strength.

Engineered resistance and risk assessment associated with insecticidal and weeds resistant transgenic cotton using wister rat model.

Stacking multiple genes into cotton crop to cop up multiple biotic stresses such as insects and weeds is a promising tool to save crop from losses. Transgenic cotton variety, VH-289, with double Bt and cp4EPSPS genes under the control of 35S promoter was used for the expression analyses and biosafety studies. The transgenic cotton plants were screened through PCR amplification of fragments, 1.7 kb for Cry1Ac, 582 bp for Cry2A and 250 bp for cp4EPSPS; which confirmed the presence of all genes transformed in transgenic cotton. The Cry1Ac + Cry2A and cp4EPSPS proteins were quantified through ELISA in transgenic cotton plants. The Glyphosate assay performed by spraying 1900 mL per acre of glyphosate Roundup further confirmed complete survival of transgenic cotton plants as compared to the non-transgenic cotton plants and all weeds. Similarly, insect infestation data determined that almost 99% insect mortality was observed in controlled field grown transgenic cotton plants as compared to the non-transgenic control plants. Evaluation of effect of temperature and soil nutrients availability on transgene expression in cotton plants was done at two different cotton growing regions, Multan and Lahore, Pakistan and results suggested that despite of higher temperature in Multan field, an increased level of Cry and cp4EPSPS proteins was recorded due to higher soil organic matter availability compared to Lahore field. Before commercialization of any transgenic variety its biosafety study is mandatory so, a 90 days biosafety study of the transgenic cotton plants with 40% transgenic cottonseeds in standard diet showed no harmful effect on wister rat model when studied for liver function, renal function and serum electrolyte.

Biochemical evidence of epicuticular wax compounds involved in cotton-whitefly interaction.

Sucking insects require a surface of plants on which the legs and the eggs of insects will adhere and to which insect mouthparts will access. The primary plant protection against insects is their surface property, which hinders the attachment of the insect's legs and eggs. The epicuticular waxes chemistry influences the fine structure of the cuticular surface. In current study, an attempt was made to investigate the variation of chemical compounds in epicuticular waxes of four cotton species that classify them resistant or susceptible i.e., Gossypium abroreum, G. hirsutum, G. arboreum wax deficient mutant (GaWM3) and G. harknessi which were evaluated for their interaction with whitefly and CLCuV transmission. Gossypium hirsutum an insect and CLCuV susceptible cotton variety, was found to have four compounds namely Trichloroacetic acid, hexadecylester, P-xylenolpthalein, 2-cyclopentene-1-ol, 1-phenyl-and Phenol, 2,5-bis [1,1- dimethyl] which could interact with chitin of whitefly while only two compounds in Gossypium arboreum an insect and CLCuV resistant cotton variety could interact with chitin of whitefly. Similarly, GaWM3 and Gossypium harkasnessi were found to have only a single compound. Number of whiteflies found on leaves of G. hirsutum was much higher as compared to other cotton species. Keeping this fact in mind a wax biosynthetic gene CER3, from Arabidopsis thaliana was transformed into G. hirsutum and the plants were evaluated for their resistance against whitefly and CLCuV transmission. In microscopic analysis transgenic plants clearly showed higher amounts of leaf waxes as compared to non-transgenics. The least whitefly population and CLCuV titer of <10,000 units was found in transgenic plants compared to non-transgenic cotton where it was ≈4.5X106 units that confirmed the role of wax in insect interaction and ultimately to CLCuV transmission. This study provides novel insight on wax related compounds involved in cotton-whitefly interaction, which potentially can help in developing more efficient control strategies for this destructive pest.

Transformation and evaluation of Broad-Spectrum insect and weedicide resistant genes in Gossypium arboreum (Desi Cotton).

Gossypium arboreum (Desi Cotton) holds a special place in cotton industry because of its inherent ability to withstand drought, salinity, and remarkable resistance to sucking pests and cotton leaf curl virus. However, it suffers yield losses due to weeds and bollworm infestation. Genetic modification of G. arboreum variety FBD-1 was attempted in the current study to combat insect and weedicide resistance by incorporating cry1Ac, cry2A and cp4-EPSPS genes under control of 35S promoter in two different cassettes using kanamycin and GUS as markers through Agrobacterium-mediated shoot apex cut method of cotton transformation. The efficiency of transformation was found to be 1.57%. Amplification of 1700 bp for cry1Ac, 167 bp for cry2A and 111 bp for cp4-EPSPS confirmed the presence of transgenes in cotton plants. The maximum mRNA expression of cry1Ac and cp4-EPSPS was observed in transgenic cotton line L3 while minimum in transgenic cotton line L1. The maximum protein concentrations of Cry1Ac, Cry2A and Cp4-EPSPS of 3.534 µg g-1, 2.534 µg g-1 and 3.58 µg-g-1 respectively were observed for transgenic cotton line L3 as compared to control cotton line. On leaf-feed-based insect bioassay, almost 99% mortality was observed for Helicoverpa armigera on the transgenic cotton plant (L3). It completely survived the 1900 ml hectare-1 glyphosate spray assay as compared to non-transgenic cotton plants. The necrotic spots appeared on the third day, leading to the complete death of control plants on the fifth day of assay. The successful multiple gene-stacking in G. arboreum FBD-1 variety could be further used for qualitative improvement of cotton fiber through plant breeding techniques.

Novel approaches to circumvent the devastating effects of pests on sugarcane.

Sugarcane (Saccharum officinarum L.) is a cash crop grown commercially for its higher amounts of sucrose, stored within the mature internodes of the stem. Numerous studies have been done for the resistance development against biotic and abiotic stresses to save the sucrose yields. Quality and yield of sugarcane production is always threatened by the damages of cane borers and weeds. In current study two problems were better addressed through the genetic modification of sugarcane for provision of resistance against insects and weedicide via the expression of two modified cane borer resistant CEMB-Cry1Ac (1.8 kb), CEMB-Cry2A (1.9 kb) and one glyphosate tolerant CEMB-GTGene (1.4 kb) genes, driven by maize Ubiquitin Promoter and nos terminator. Insect Bio-toxicity assays were carried out for the assessment of Cry proteins through mortality percent of shoot borer Chilo infuscatellus at 2nd instar larvae stage. During V0, V1 and V2 generations young leaves from the transgenic sugarcane plants were collected at plant age of 20, 40, 60, 80 days and fed to the Chilo infuscatellus larvae. Up to 100% mortality of Chilo infuscatellus from 80 days old transgenic plants of V2 generation indicated that these transgenic plants were highly resistant against shoot borer and the gene expression level is sufficient to provide complete resistance against target pests. Glyphosate spray assay was carried out for complete removal of weeds. In V1-generation, 70-76% transgenic sugarcane plants were found tolerant against glyphosate spray (3000 mL/ha) under field conditions. While in V2-generation, the replicates of five selected lines 4L/2, 5L/5, 6L/5, L8/4, and L9/6 were found 100% tolerant against 3000 mL/ha glyphosate spray. It is evident from current study that CEMB-GTGene, CEMB-Cry1Ac and CEMB-Cry2A genes expression in sugarcane variety CPF-246 showed an efficient resistance against cane borers (Chilo infuscatellus) and was also highly tolerant against glyphosate spray. The selected transgenic sugarcane lines showed sustainable resistance against cane borer and glyphosate spray can be further exploited at farmer's field level after fulfilling the biosafety requirements to boost the sugarcane production in the country.