Construction of an Infectious Clone of the Badnavirus Cacao Swollen Shoot Ghana M Virus and Infectivity by Gene Gun- and Agrobacterium-Mediated Inoculation

Cacao swollen shoot disease (CSSD) is a damaging disease of Theobroma cacao L. associated with infection by a group of poorly characterized badnaviral species. To establish causality and characterize the symptomatology associated with infection by the badnavirus cacao swollen shoot Ghana M virus (CSSGMV), an infectious clone (1.3-mer) was constructed and used to inoculated cacao “Amelonado” seedlings by biolistic inoculation (BI; n = 18) and agroinoculation (AI; n = 15). Newly expanded leaves of BI (10/18) and AI (12/15) plants developed foliar mosaic and curling symptoms 30-days post inoculation (dpi), with chlorotic mottling and necrotic crinkling being evident by 90 dpi. By 120 dpi, three of 15 AI plants exhibited characteristic stem-swelling. Viral infection was verified by PCR-amplification and sequencing of a 1068 bp fragment of the CSSGMV ORF3 from newly expanding leaves 60 dpi. The PCR results indicated that 14 of 18 and 15 of 15 BI and AI plants, respectively, were systemically infected. The complete CSSGMV genome sequence was determined, by Illumina sequencing, from representative AI and BI plants and shared >99.5% pairwise nucleotide identity with CSSGMV-Nig9 (GenBank Accession No. MH785299). Based on the development of characteristic CSSD symptoms and recovery of partial and complete genome sequences of CSSGMV-Nig9 from systemically infected cacao plants, Koch's postulates have been fulfilled.

A discussion on cotton transformation during the last decade (2010–2021); an update on present trends and future prospects

The introduction of genetically modified (GM) cotton in 1996 in the US and its worldwide spread later rejuvenated cotton production in many parts of the world. The evolution is continued since then and currently, the 3rd and fourth generation of same GM cotton is grown in many parts of the world. The GM cotton introduced in 1996 was simple Bt cotton that expressed a single Cry1Ac gene, the later generation carried multiple Cry genes along with the genes controlling herbicide tolerance. Current day GM cotton does not only give stable resistance against lepidopteran insects but also facilitates the farmers to spray broad-spectrum herbicides without harming the crop. The evolution of GM cotton is continued both on the basic and applied side and interventions have been introduced during the last decade. Earlier the cotton transformation was limited to Cocker strains which are getting possible in many other varieties, too. It is successful with both gene gun, and Agrobacterium and inplanta transformation has made it a routine activity. Apart from overexpression studies for various purposes including biotic, abiotic, and quality traits, RNAi and genome editing are explored vigorously. Through this review, we have tried to explore and discuss various interventions for improving transformation protocols, the applications of cotton transformation, and future strategies being developed to get maximum benefits from this technology during the last decade.

A Simple and Efficient Genetic Immunization Protocol for the Production of Highly Specific Polyclonal and Monoclonal Antibodies against the Native Form of Mammalian Proteins

We have generated polyclonal and monoclonal antibodies by genetic immunization over the last two decades. In this paper, we present our most successful methodology acquired over these years and present the animals in which we obtained the highest rates of success. The technique presented is convenient, easy, affordable, and generates antibodies against mammalian proteins in their native form. This protocol requires neither expensive equipment, such as a gene gun, nor sophisticated techniques such as the conjugation of gold microspheres, electroporation, or surgery to inject in lymph nodes. The protocol presented uses simply the purified plasmid expressing the protein of interest under a strong promoter, which is injected at intramuscular and intradermal sites. This technique was tested in five species. Guinea pigs were the animals of choice for the production of polyclonal antibodies. Monoclonal antibodies could be generated in mice by giving, as a last injection, a suspension of transfected cells. The antibodies detected their antigens in their native forms. They were highly specific with very low non-specific background levels, as assessed by immune-blots, immunocytochemistry, immunohistochemistry and flow cytometry. We present herein a detailed and simple procedure to successfully raise specific antibodies against native proteins.

Genetic Transformation Methods for Crop Improvement- A Brief Review

Decades of documented successful pieces of evidence in agritech have shown us a clear picture of the importance of biotechnology in crop improvement. The production in agriculture should have steady growth and to achieve these objective conventional methods should go on parallel with biotechnological approaches. Genetic engineering which has revolutionized the path of crop improvement involves the identification and transfer of novel genes into the existing elite cultivars. Different methods of transferring the gene into plant cells have been developed and continuous efforts have been made to increase its efficiency. Both direct and indirect method of gene transfer has its own merits and demerits. Efforts have been made continuously to eliminate drawbacks and to develop an easy, elite and eco-friendly method to transfer genes. The transformation method which is a base of genetic engineering is vital and Agrobacterium-mediated gene transfer and gene gun have shown to be doing well in recent years. As a whole the methodology involved, merits and demerits of different methods have been briefly discussed.

Creation of Cultures Containing Mutations Linked with Cardiovascular Diseases using Transfection and Genome Editing

Objective: In this review article, we analyzed the literature on the creation of cultures containing mutations associated with cardiovascular diseases (CVD) using transfection, transduction and editing of the human genome. Methods: We described different methods of transfection, transduction and editing of the human genome, used in the literature. Results: We reviewed the researches in which the creation of сell cultures containing mutations was described. According to the literature, system CRISPR/Cas9 proved to be the most preferred method for editing the genome. We found rather promising and interesting a practically undeveloped direction of mitochondria transfection using a gene gun. Such a gun can direct a genetically-engineered construct containing human DNA mutations to the mitochondria using heavy metal particles. However, in human molecular genetics, the transfection method using a gene gun is unfairly forgotten and is almost never used. : Ethical problems arising from editing the human genome were also discussed in our review. We came to a conclusion that it is impossible to stop scientific and technical progress. It is important that the editing of the genome takes place under the strict control of society and does not bear dangerous consequences for humanity. To achieve this, the constant interaction of science with society, culture and business is necessary. Conclusion: he most promising methods for the creation of cell cultures containing mutations linked with cardiovascular diseases, were system CRISPR/Cas9 and the gene gun.

Wheat dwarf virus infectious clones allow to infect wheat and Triticum monococcum plants

We constructed Wheat dwarf virus (WDV) infectious clones in the bacterial plasmids pUC18 and pIPKb002 and tested their ability to inoculate plants using Bio-Rad Helios Gene Gun biolistic inoculation method and Agrobacterium tumefaciens agroinoculation method, and we then compared them with the natural inoculation method via viruliferous P. alienus. Infected plants were generated using both infectious clones, whereas the agroinoculation method was able to produce strong systemic infection in all three tested cultivars of wheat and Triticum monococcum, comparable to plants inoculated by viruliferous P. alienus. Infection was confirmed by DAS-ELISA, and WDV titres were quantified using qPCR. The levels of remaining bacterial plasmid DNA were also confirmed to be zero.