Agrobacterium-Mediated Transformation for the Development of Transgenic Crops; Present and Future Prospects.

Plant transformation based on Agrobacterium-mediated transformation is a technique that mimics the natural agrobacterium system for gene(s) introduction into crops. Through this technique, various crop species have been improved/modified for different trait/s, showing a successful genetic transformation so far. This technique has many advantages over other transformation methods such as stable integration of transgene, cost effective. However, there are many limitations of this technology such as mostly the crops are recalcitrant to agrobacterium, low transformation efficiency, transgene integration as well as off targets. So, it's very important to explore the major limitations and possible solutions for Agrobacterium-mediated transformation in order to increase its genetic transformation efficiency. Therefore, the present review article gives a comprehensive study how the transgenic crops are developed using Agrobacterium-mediated transformation, crops that have already been modified through this method, and risks associated with transgenic plants based on Agrobacterium-mediated transformation. Moreover, the challenges and problems associated with Agrobacterium-mediated transformation and how those problems can be solved in future for a successful genetic transformation of crops using modern biotechnology techniques such as CRISPR/Cas9 systems. The present review article will be really helpful for the audience those working on Genome editing of crops using Agrobacterium-mediated transformation and will opens many ways for future plant genetic transformation.

Improvement of Soybean; A Way Forward Transition from Genetic Engineering to New Plant Breeding Technologies.

Soybean is considered one of the important crops among legumes. Due to high nutritional contents in seed (proteins, sugars, oil, fatty acids, and amino acids), soybean is used globally for food, feed, and fuel. The primary consumption of soybean is vegetable oil and feed for chickens and livestock. Apart from this, soybean benefits soil fertility by fixing atmospheric nitrogen through root nodular bacteria. While conventional breeding is practiced for soybean improvement, with the advent of new biotechnological methods scientists have also engineered soybean to improve different traits (herbicide, insect, and disease resistance) to fulfill consumer requirements and to meet the global food deficiency. Genetic engineering (GE) techniques such as transgenesis and gene silencing help to minimize the risks and increase the adaptability of soybean. Recently, new plant breeding technologies (NPBTs) emerged such as zinc-finger nucleases, transcription activator-like effector nucleases, and Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR/Cas9), which paved the way for enhanced genetic modification of soybean. These NPBTs have the potential to improve soybean via gene functional characterization precision genome engineering for trait improvement. Importantly, these NPBTs address the ethical and public acceptance issues related to genetic modifications and transgenesis in soybean. In the present review, we summarized the improvement of soybean through GE and NPBTs. The valuable traits that have been improved through GE for different constraints have been discussed. Moreover, the traits that have been improved through NPBTs and potential targets for soybean improvements via NPBTs and solutions for ethical and public acceptance are also presented.

Plant Virus-Derived Vectors for Plant Genome Engineering.

Advances in genome engineering (GE) tools based on sequence-specific programmable nucleases have revolutionized precise genome editing in plants. However, only the traditional approaches are used to deliver these GE reagents, which mostly rely on Agrobacterium-mediated transformation or particle bombardment. These techniques have been successfully used for the past decades for the genetic engineering of plants with some limitations relating to lengthy time-taking protocols and transgenes integration-related regulatory concerns. Nevertheless, in the era of climate change, we require certain faster protocols for developing climate-smart resilient crops through GE to deal with global food security. Therefore, some alternative approaches are needed to robustly deliver the GE reagents. In this case, the plant viral vectors could be an excellent option for the delivery of GE reagents because they are efficient, effective, and precise. Additionally, these are autonomously replicating and considered as natural specialists for transient delivery. In the present review, we have discussed the potential use of these plant viral vectors for the efficient delivery of GE reagents. We have further described the different plant viral vectors, such as DNA and RNA viruses, which have been used as efficient gene targeting systems in model plants, and in other important crops including potato, tomato, wheat, and rice. The achievements gained so far in the use of viral vectors as a carrier for GE reagent delivery are depicted along with the benefits and limitations of each viral vector. Moreover, recent advances have been explored in employing viral vectors for GE and adapting this technology for future research.

A source of resistance against yellow mosaic disease in soybeans correlates with a novel mutation in a resistance gene.

Yellow mosaic disease (YMD) is one of the major devastating constraints to soybean production in Pakistan. In the present study, we report the identification of resistant soybean germplasm and a novel mutation linked with disease susceptibility. Diverse soybean germplasm were screened to identify YMD-resistant lines under natural field conditions during 2016-2020. The severity of YMD was recorded based on symptoms and was grouped according to the disease rating scale, which ranges from 0 to 5, and named as highly resistant (HR), moderately resistant (MR), resistant (R), susceptible (S), moderately susceptible (MS), and highly susceptible (HS), respectively. A HR plant named "NBG-SG Soybean" was identified, which showed stable resistance for 5 years (2016-2020) at the experimental field of the National Institute for Biotechnology and Genetic Engineering (NIBGE), Faisalabad, Pakistan, a location that is a hot spot area for virus infection. HS soybean germplasm were also identified as NBG-47 (PI628963), NBG-117 (PI548655), SPS-C1 (PI553045), SPS-C9 (PI639187), and cv. NARC-2021. The YMD adversely affected the yield and a significant difference was found in the potential yield of NBG-SG-soybean (3.46 ± 0.13a t/ha) with HS soybean germplasm NARC-2021 (0.44 ± 0.01c t/ha) and NBG-117 (1.12 ± 0.01d t/ha), respectively. The YMD incidence was also measured each year (2016-2020) and data showed a significant difference in the percent disease incidence in the year 2016 and 2018 and a decrease after 2019 when resistant lines were planted. The resistance in NBG-SG soybean was further confirmed by testing for an already known mutation (SNP at 149th position) for YMD in the Glyma.18G025100 gene of soybean. The susceptible soybean germplasm in the field was found positive for the said mutation. Moreover, an ortholog of the CYR-1 viral resistance gene from black gram was identified in soybean as Glyma.13G194500, which has a novel deletion (28bp/90bp) in the 5`UTR of susceptible germplasm. The characterized soybean lines from this study will assist in starting soybean breeding programs for YMD resistance. This is the first study regarding screening and molecular analysis of soybean germplasm for YMD resistance.

Circular DNA enrichment sequencing reveals the viral/satellites genetic diversity associated with the third epidemic of cotton leaf curl disease.

Cotton leaf curl disease (CLCuD) is the most important limiting factor for cotton production in Pakistan. The CLCuD passed through two major epidemics in this region with distinct begomoviruses/satellites complexes. Since 2015 the disease has again started to appear in epidemic form, causing heavy losses to cotton crop, which we termed as the "third epidemic". We applied CIDER-seq (Circular DNA Enrichment Sequencing), a recently developed sequencing method for PCR-free virus enrichment to produce a full length read of a single circular viral genome coupled with Sanger sequencing to explore the genetic diversity of the disease complex. We identified a highly recombinant strain of Cotton leaf curl Multan virus and a recently evolved strain of Cotton leaf curl Multan betasatellite that are dominant in all major cotton growing regions in the country. Moreover, we also identified multiple species of alphasatellites with one distinct species, Mesta yellow vein mosaic alphasatellite (MeYVMA) for the first time in cotton. Relative abundance of virus and associated satellites was also determined by real-time quantitative PCR. To the best of our knowledge, this is the first study that determined the CLCuD complex associated with its third epidemic.

Application of Zinc and Iron-Based Fertilizers Improves the Growth Attributes, Productivity, and Grain Quality of Two Wheat (Triticum aestivum) Cultivars.

Field-based experiments were conducted during wheat cultivation seasons of 2017-2018 and 2018-2019 to minimize the impact of hidden hunger (micronutrient deficiencies) through agronomic biofortification of two wheat cultivars with zinc and iron. Two spring-planted bread wheat cultivars: Zincol-16 (Zn-efficient) and Anaj-17 (Zn-inefficient with high-yield potential) were treated with either zinc (10 kg/ha), iron (12 kg/ha), or their combination to study their effect on some growth attributes (plant height, tillers, and spike length, etc.,), productivity, and quality. No application of zinc and iron or their combinations served as the control. Maximum Zn and Fe contents of grains were improved by sole application of Zn and Fe, respectively. A higher concentration of Ca in grains was observed by the combined application of Zn and Fe. Starch contents were found maximum by sole application of Fe. Sole or combined application of Zn and Fe reduced wet gluten contents. Maximum proteins were recorded in Anaj-17 under control treatments. Zincol-16 produced maximum ionic concentration, starch contents, and wet gluten as compared to Anaj-17. Yield and growth attributes were also significantly (p < 0.05) improved by combined application as compared to the sole application of Zn or Fe. The combined application also produced the highest biological and grain yield with a maximum harvest index. Cultivar Anaj-17 was found more responsive regarding growth and yield attributes comparatively. The findings of the present study showed that the combined application of Zn and Fe produced good quality grains (more Zn, Fe, Ca, starch, and less gluten concentrations) with a maximum productivity of bread wheat cultivars.

Protective effects of dietary glycine and glutamic acid toward the toxic effects of oxidized mustard oil in rabbits.

The protective role of glycine and glutamic acid against the toxic effects of oxidized oil was studied for the first time. Mustard seed oil was thermally oxidized and characterized for quality characteristics and polyphenolic composition using reversed phase HPLC-DAD. Significant changes in the quality characteristics occurred with thermal oxidation. Fourteen polyphenolic compounds were identified and quantified in oils. Quercetin-3-glucoside, quercetin-3-feruloylsophoroside, catechin, quercetin-3-rutinoside, quercetin-3,7-diglucoside, sinapic acid and vanillic acid hexoside were the major compounds in the fresh and oxidized oil. Oxidized, un-oxidized mustard oils, glycine and glutamic acid were given to rabbits alone or in combination. The biochemical responses were studied in terms of haematological and biochemical parameters and histopathology. It has been observed that biochemical and haematological parameters were adversely affected by the oxidized oil, while supplementation of both amino acids was beneficial in normalizing these parameters. Both amino acids alone have no significant effects, however, oxidized oil affected the liver by enhancing fat accumulation, causing hepatitis, reactive Kupffer cells and necrosis. The co-administration of oxidized oils with glycine or glutamic acid revealed significant recovery of the liver structure and function. In conclusion, glycine or glutamic acid is beneficial and protective against food toxicity and can be considered as an ameliorative food supplement.