Evaluation of two cotton varieties CRSP1 and CRSP2 for genetic transformation efficiency, expression of transgenes Cry1Ac+Cry2A, GT gene and insect mortality

AbstractExpression of the transgene with a desirable character in crop plant is the ultimate goal of transgenic research. Transformation of two Bt genes namely Cry1Ac and Cry2A cloned as separate cassette under 35S promoter in pKHG4 plant expression vector was done by using shoot apex cut method of Agrobacterium. Molecular confirmation of putative transgenic cotton plants for Cry1Ac, Cry2A and GT gene was done through PCR and ELISA. Transformation efficiency of CRSP-1 and CRSP-2 was calculated to be 1.2 and 0.8% for Cry1Ac while 0.9 and 0.6% for Cry2A and 1.5 and 0.7% for GTG respectively. CRSP-1 was found to adopt natural environment (acclimatized) earlier than CRSP-2 when exposed to sunlight for one month. Expression of Cry1Ac, Cry2A and GTG was found to be 1.2, 1 and 1.3ng/μl respectively for CRSP-1 as compared to CRSP-2 where expression was recorded to be 0.9, 0.5 and 0.9ng/μl respectively. FISH analysis of the transgenic CRSP-1 and CRSP-2 demonstrated the presence of one and two copy numbers respectively. Similarly, the response of CRSP-1 against Glyphosate @1900ml/acre was far better with almost negligible necrotic spot and efficient growth after spray as compared to CRSP-2 where some plants were found to have necrosis and negative control where the complete decay of plant was observed after seven days of spray assay. Similarly, almost 100% mortality of 2nd instar larvae of Heliothis armigera was recorded after three days in CRSP-1 as compared CRSP-2 where insect mortality was found to be less than 90%. Quantitatively speaking non transgenic plants were found with 23–90% leaf damage by insect, while CRSP-1 was with less than 5% and CRSP-2 with 17%. Taken together CRSP1 was found to have better insect control and weedicide resistance along with its natural ability of genetic modification and can be employed by the valuable farmers for better insect control and simultaneously for better production

Plant genome editing using engineered nucleases and success of CRISPR/Cas9 system

Development of new plant breeding techniques have facilitated easy manipulation of plants at genetic level. Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/CRISPR associated protein9 (Cas9) system is a valuable addition in programmable nucleases. The CRISPR/Cas9 system uses an RNA component to recognize a target DNA sequences and it has shown promising results with respect to simultaneous editing of multigenic plant traits. In this review, components of CRISPR/Cas9, their construction and its methods of delivery to plant cells are analyzed. Variation in nucleotide sequence of the protospacer adjacent motif, codon optimization and progress in web-based bioinformatic tools, will make CRISPR/Cas9 systems more efficient for plants. Development and optimization of protocols to efficiently target all plant species is still under development. Along with this, methods to inspect induced mutation and efficiency of the system have also been reviewed. Auxiliary improvements and understanding are still required to expand the CRISPR/Cas9 systems to target complex genome architectures and epigenetic elements

Genetic effects of Calotropis procera CpTIP1 gene on fiber quality in cotton (Gossypium hirsutum)

Background: The importance of cotton crop (Gossypium hirsutum) in textile industry is based on its fiber quality. A number of fiber-specific genes play important role in the development of cotton fiber. Previous studies have demonstrated the importance of genes that are responsible for metabolic functions and their involvement in cotton fiber development.Methods: This study was focused at successful Agrobacterium mediated stable transformation of the fiber gene CpTIP1, isolated from the wild plant Calotropis procera, into cotton variety NIAB-846 for one generation. Results: Transformation efficiency was calculated to be 1.01% for the target gene. Different molecular techniques such as PCR were used for confirmation and Real-Time PCR was used to check the level of quantitative expression of fiber expansin gene in putative transgenic cotton plants. On the base of molecular analysis, results showed higher expression level of fiber gene (CpTIP1) in transgenic plants as compared to the control plants.Conclusion: The results of this study support the idea of improved cotton fiber through genetic modification especially the cotton fiber strength

Defense strategies of cotton against whitefly transmitted CLCuV and Begomoviruses

Cotton leaf curl virus (CLCuV) is economically important monopartite Geminivirus which is transmitted by whitefly in persistent circulative manner. In Pakistan, CLCuV causes severe damage to Gossypium hirsutum whereas G. arboreum is resistant to this virus. A total of 86 articles were included in this study that were searched through web of knowledge, web of science and google scholar by using the keywords of cotton, CLCuV, waxes, Begomoviruses and transgenic techniques used against Begomoviruses in plants. Various transgenic strategies i.e., pathogen derived resistance (with and without protein expression) and non - pathogen derived resistance have been adopted to control this virus or its vector. Beside these, some natural defense mechanisms of plant also protect it against the vector. The cuticular waxes make the insects’ attachment difficult to plant surfaces and act as a physical barrier. The cuticular waxes in G. arboreum act as first line of defense against whiteflies and thus CLCuV. Some other defense strategies may involve hindering the insect movements or depriving it from food due to thick waxy layer. Biotechnological strategies against various Begomoviruses including CLCuV were found successful in some crops except cotton. Whereas, the natural defense strategies in G. arboreum i.e., long trichome or presence of inorganic salts with increased concentration of waxes, provide good defense strategy against whiteflies, CLCuV and other pathogens

Science Behind Cotton Transformation

The introduction of foreign genes into plant has made possible to bring out desired traits into crop of our own interest. With the advancement in cell biology, regeneration of plants from single cell and advent of different procedures for gene transformation to the plants have opened new avenues for the efficient and applicable implementation of biotechnology for the modifications of desired crop characteristics. Identifications and isolation of different genes for various traits from different organisms have made possible to get the crop plants with modified characters. Over time improvement has been made in transformation technology depending upon the crop of interest. The efficiency of plant transformation has been increased with advances in plant transformation vectors and methodologies, which resulted in the improvement of crops. A detailed discussion on advanced techniques for genetic modification of plants with their handy use and limitation has been focused in this chapter

Dissemination of Bt cotton in cotton growing belt of Pakistan

AbstractBackground: Adoptability of biotech crops has climbed up dramatically in the world from 1.7 million hectares in its introductory period in 1996 to 170 million hectare in 2012. Area planted to biotech crop increased to over one-fourth of the world total in 2005-06. The data collected also demonstrates that developing countries like Pakistan are making tremendous development in production of Biotech crops especially cotton.Methodology: Random samples have been collected and received from different cotton growing areas of Pakistan from 2007 to date for Bt gene confirmation and expression. Genomic DNA was isolated and analyzed through internal reference primers from SadI gene of cotton genome. Samples were analyzed by PCR for detection of Bt genes including CEMB Cry1Ac+2A double Bt gene. ELISA was done for confirmation of Bt protein by using Envirologix Quantiplate ELISA kit Cry1Ab/Ac Cat # AP003 and Quantiplate ELISA kit Cry2A Cat # AP005 according to manufacturer instruction.Results: Development of Bt Cotton in Pakistan is as old as in advanced countries of the world. Pakistan has become the fourth country of the world for production of indigenous Bt cotton by utilizing their local cotton varieties for genetic modifications. The support of various documentary proofs like research articles, patents, projects, Ph. D and M.Phil studies generated by Centre of Excellence in Molecular Biology (CEMB) greatly strengthen the data of Bt cotton development in Pakistan. Pakistan Central Cotton Committee (PCCC)declared locally developed Bt cotton as the best performing of all over Pakistan among other international product.Conclusion: Progress made in development of indigenous Bt cotton variety by CEMB Pakistan and their excellent performance in field determined their increased adoptability ratio in farmers. The formal approval process which is going to be completed in near future will open the doors for farmers and breeders to utilize this material for better economy of Pakistan

Genomics of Salinity Tolerance in Plants

Plants are frequently exposed to wide range of harsh environmental factors, such as drought, salinity, cold, heat, and insect attack. Being sessile in nature, plants have developed different strategies to adapt and grow under rapidly changing environments. These strategies involve rearrangements at the molecular level starting from transcription, regulation of mRNA processing, translation, and protein modification or its turnover. Plants show stress-specific regulation of transcription that affects their transcriptome under stress conditions. The transcriptionally regulated genes have different roles under stress response. Generally, seedling and reproductive stages are more susceptible to stress. Thus, stress response studies during these growth stages reveal novel differentially regulated genes or proteins with important functions in plant stress adaptation. Exploiting the functional genomics and bioinformatics studies paved the way in understanding the relationship between genotype and phenotype of an organism suffering from environmental stress. Future research programs can be focused on the development of transgenic plants with enhanced stress tolerance in field conditions based upon the outcome of genomic approaches and knowing the mystery of nucleotides sequences hidden in cells

Trackable CEMB-Klean Cotton Transgenic Technology: Affordable Climate Neutral Agri-biotech Industrialization for Developing Countries

Background: Transgenic technology reflects the incorporation of novel useful traits in crop plants like cotton for economic benefits by overcoming the problems including insects’ pests and weeds in special. The present study is the success story of the continuous effort of CEMB team started back in the 1990s.Methods: This study includes characterization of a large number of Bacillus thuringiensis (Bt) strains taken from local soil and subjected to direct transformation of isolated BT genes into local cotton cultivars. Protocols for transformation into cotton plants were optimized and validated by the development of double gene codon optimized (Cry1Ac and Cry2A) transgenic cotton varieties.Results: The resulting GMOs in the form of CEMB-33, CA-12, CEMB-66 have been approved by Punjab Seed Council in 2013 and 2016 respectively. Double Bt and weedicide resistant cotton harboring CEMB-Modified and codon optimized cp4EPSPS (GTGene). These varieties can tolerate glyphosate spray @ 1900ml per acre without the appearance of necrotic spots/shedding and complete removal of all surrounding weeds in the cotton field is a significant advance to boost cotton production without spending much on insecticides and herbicides.Conclusion: In the current report, two unique sets of primers which amplify 1.1 Kb for CEMB-double Bt genes and 660 bp product for CEMB-Modified cp4EPSPS (GTGene) were tested. CEMB cotton variety CKC-01 is specially designed as low cost and easy to use by local farmer’s technology has the potential to revolutionize the cotton growing culture of the country.Keywords: Event detection; Bt Cotton; CEMB transgenic technology; GTGen