Maize Chlorotic Mottle Virus (MCMV) is a deleterious pathogen which causes Maize Lethal Necrosis Disease that result in substantial yield loss of Maize crop worldwide. The positive-sense RNA genome of MCMV (4.4 kb) encodes six proteins: P32 (32kDa protein), RNA dependent RNA polymerases (P50 and P111), P31 (31kDa protein), P7 (7kDa protein), coat protein (25 kDa). P31, P7 and coat protein are encoded from sgRNA1, located at the 3′end of the genome and sgRNA2, located at the extremity of the 3′genome end. The objective of this study is to locate the possible attachment sites of Zea mays derived miRNAs in the genome of MCMV, using four diverse efficient miRNA target prediction algorithms. In total, 321 mature miRNAs were retrieved from miRBase (miRNA database) and were tested for hybridization of MCMV. These algorithms considered the parameters of seed pairing, minimum free energy, target site accessibility, multiple target sites, pattern recognition and folding energy for attachment. Out of 321 only 10 most efficient maize miRNAs are predicted for silencing of MCMV genome. The results of this study can hence act as the first step towards the development of MCMV resistant transgenic Maize plants through expression of the selected miRNAs.

More than 50 countries around the globe cultivate cotton on a large scale. It is a major cash crop of Pakistan and is considered “white gold” because it is highly important to the economy of Pakistan. In addition to its importance, cotton cultivation faces several problems, such as insect pests, weeds, and viruses. In the past, insects have been controlled by insecticides, but this method caused a severe loss to the economy. However, conventional breeding methods have provided considerable breakthroughs in the improvement of cotton, but it also has several limitations. In comparison with conventional methods, biotechnology has the potential to create genetically modified plants that are environmentally safe and economically viable. In this study, a local cotton variety VH 289 was transformed with two Bt genes (Cry1Ac and Cry2A) and a herbicide resistant gene (cp4 EPSPS) using the Agrobacterium mediated transformation method. The constitutive CaMV 35S promoter was attached to the genes taken from Bacillus thuringiensis (Bt) and to an herbicide resistant gene during cloning, and this promoter was used for the expression of the genes in cotton plants. This construct was used to develop the Glyphosate Tolerance Gene (GTGene) for herbicide tolerance and insecticidal gene (Cry1Ac and Cry2A) for insect tolerance in the cotton variety VH 289. The transgenic cotton variety performed 85% better compared with the non-transgenic variety. The study results suggest that farmers should use the transgenic cotton variety for general cultivation to improve the production of cotton.

Potato virus Y has emerged as a threatening problem in all potato growing areas around the globe PVY reduces the yield and quality of potato cultivars. During last 30 years, significant genetic changes in PVY strains have been observed with an increased incidence associated with crop damage. In the current study, computational approaches were applied to predict Potato derived miRNA targets in PVY genome. PVY genome is about 9 thousand nucleotides approximately which transcribes 6 genes CI, NIa, NIb-Pro, HC-Pro, CP and VPg. A total of 343 mature miRNAs were retrieved from miRbase database and searched for their target sequences in PVY genes using minimum free energy (mfe), minimum folding energy, sequence complementarity and mRNA-miRNA hybridization approaches. Identified Potato miRNAs against viral mRNA targets have antiviral activities leading to either translational inhibition by mRNA cleavage/mRNA blockage or both. We have found 86 miRNAs targeting PVY genome at 151 different sites on PVY genome. Moreover, only 36 miRNA potentially targeted the PVY genome at 101 loci. CI gene of PVY genome was targeted by 32 miRNAs followed by complementarity by 26, 19, 18, 16 and 13 miRNAs respectively. Most importantly, we found 5 miRNAs (miR160a-5p, miR7997b, miR166c-3p, miR399h and miR5303d) could target CI, NIa, NIb-Pro, HC-Pro, CP and VPg genes of PVY. The predicted miRNAs can be used for development of PVY resistant potato crops in future.

Sugarcane bacilliform virus (SCBV) is considered one of the most economically damaging pathogens for sugarcane production worldwide. Three open reading frames (ORFs) are characterized in the circular, ds-DNA genome of the SCBV; these encode for a hypothetical protein (ORF1), a DNA binding protein (ORF2), and a polyprotein (ORF3). A comprehensive evaluation of sugarcane (Saccharum officinarum L.) miRNAs for the silencing of the SCBV genome using in silico algorithms were carried out in the present study using mature sugarcane miRNAs. miRNAs of sugarcane are retrieved from the miRBase database and assessed in terms of hybridization with the SCBV genome. A total of 14 potential candidate miRNAs from sugarcane were screened out by all used algorithms used for the silencing of SCBV. The consensus of three algorithms predicted the hybridization site of sof-miR159e at common locus 5534. miRNA–mRNA interactions were estimated by computing the free-energy of the miRNA–mRNA duplex using the RNAcofold algorithm. A regulatory network of predicted candidate miRNAs of sugarcane with SCBV—ORFs, generated using Circos—is used to identify novel targets. The predicted data provide useful information for the development of SCBV-resistant sugarcane plants.

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; GTGene