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

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

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

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

Selection of potent bacterial strain for over-production of PHB by using low cost carbon source for eco-friendly bioplastics

Background: The microbial PHB production is a promising tool for the plastic industry for the synthesis of environmental friendly, biodegradable plastic in contrast to the conventional petro-chemical based non-degradable plastics. The selection of potent bacterial strains, inexpensive carbon source, efficient fermentation and recovery processes are important aspects that were taken into account during this study. Methods: Different bacterial strains i.e. Bacillus Spp, P. putida and P. fluorescens were screened for maximum PHB production. Under media optimization, various carbon and nitrogen sources (alone or in combination) were used to achieve the maximum PHB production. Finally the degradation tests of the PHB sheet were also performed to test its biodegradability potential. Results: Shake flask studies have shown the PHB concentrations upto 7.02, 4.50 and 34.4 mg/g of dry cell mass of P. putida, P. fluorescens and Bacillus Spp. respectively. Almost same results were observed at laboratory scale production of PHB in 10 L fermenter i.e. 6.28, 6.23 and 39.5 mg/g of dry cell mass by P. putida, P. fluorescens and Bacillus Spp. respectively. On the basis of these observations, Bacillus Spp. was chosen for laboratory scale PHB production. Corn steep liquor (4%) was chosen as the best medium to achieve the highest PHB contents. Isolated PHB has shown biodegradation in soil up to 86.7% at 37oC. Conclusion: The Bacillus Spp. Proved to be the best strain for PHB production on only 4% CSL which is cheapest and easily available

The Myth of Aloe vera phytochemicals and its advancement as medicinal and plant packed cosmetics

A succulent plant Aloe vera has been extensively utilized for various pharmaceutical and biomedical products. Its bioactive components that have different properties of anti-inflammation, anti-oxidation, anti-microbial and immunomodulation have made it more attractive for the scientific community to further explore its hidden facts about its utilization and also in addition to improve its properties through expression of some useful protein which can make it complete plant packed cosmetics. This study briefly summarizes fundamental active components, their biological activities, and significant applications of the constituents found in Aloe vera, and extensively surveyed its research progress in cytology and molecular biology with an emphasis on genetic engineering. Aloe constituents are studied in the context of therapeutics, cosmetics, and hydration, antibacterial and antiviral applications. The information that has been presented here is useful for better comprehending and investigating its possible therapeutic and culinary applications and provides routes for future studies.&nbsp