Selection of large quantities of embryogenic calli from indica rice seeds for production of fertile transgenic plants using the biolistic method

The microprojectile bombardment of immature embryos has proven to be effective in transforming many indica rice varieties. One of the drawbacks of using immature embryos is the requirement of a large number of high quality immature embryos, which itself is a tedious and laborious process. To circumvent these problems, we have developed a procedure, using indica variety TN1 as a model that generates highly homogenous populations of embryogenic subcultured calli by selectively propagating a small number of regeneration-proficient calli derived from seeds. Thousands of embryogenic calli were produced from 50 seeds within 10 weeks. Ten to 20 independent R0 transgenic lines were regenerated per 500 embryogenic calli bombarded. The convenience and reliability offered by this transformation system has made transformation of indica rice a routine procedure.Abbreviations 2,4-D 2,4-dichlorophenoxy acetic acid - NAA naphthalene acetic acid - BAP 6-benzylaminopurine - kb kilobase - GUS -glucuronidase - X-gluc 5-bromo-4-chloro-3-indolyl--D-glucuronide - HPH hygromycin B phosphotransferase     

Resistance to wheat streak mosaic virus in transgenic wheat engineered with the viral coat protein gene

Wheat (Triticum aestivum) plants were stably transformed with the coat protein (CP) gene of wheat streak mosaic virus (WSMV) by the biolistic method. Eleven independently transformed plant lines were obtained and five were analyzed for gene expression and resistance to WSMV. One line showed high resistance to inoculations of two WSMV strains. This line had milder symptoms and lower virus titer than control plants after inoculation. After infection, new growth did not show symptoms. The observed resistance was similar to the recovery type resistance described previously using WSMV NIb transgene and in other systems. This line looked morphologically normal but had an unusually high transgene copy number (approximately 90 copies per 2C homozygous genome). Northern hybridization analysis indicated a high level of degraded CP mRNA expression. However, no coat protein expression was detected.     

Activity of the 5′ regulatory regions of the rice polyubiquitin <Emphasis Type="Italic">rubi3</Emphasis> gene in transgenic rice plants as analyzed by both <Emphasis Type="Italic">GUS</Emphasis> and <Emphasis Type="Italic">GFP</Emphasis> reporter genes

Ubiquitin is an abundant protein involved in protein degradation and cell cycle control in plants and rubi3 is a polyubiquitin gene isolated from rice (Oryza sativa L.). Using both GFP and GUS as reporter genes, we analyzed the expression pattern of the rubi3 promoter as well as the effects of the rubi3 5'-UTR (5' untranslated region) intron and the 5' terminal 27 bp of the rubi3 coding sequence on the activity of the promoter in transgenic rice plants. The rubi3 promoter with the 5'-UTR intron was active in all the tissue and cell types examined and supported more constitutive expression of reporter genes than the maize Ubi-1 promoter. The rubi3 5'-UTR intron mediated enhancement on the activity of its promoter in a tissue-specific manner but did not alter its overall expression pattern. The enhancement was particularly intense in roots, pollen grains, inner tissue of ovaries, and embryos and aleurone layers in maturing seeds. The translational fusion of the first 27 bp of the rubi3 coding sequence to GUS gene further enhanced GUS expression directed by the rubi3 promoter in all the tissues examined. The rubi3 promoter should be an important addition to the arsenal of strong and constitutive promoters for monocot transformation and biotechnology.     

Biological control ofFusarium oxysporum f.sp.cubense in banana by inoculation withPseudomonas fluorescens

Native strains ofPseudomonas fluorescens exhibitedin vitro antibiosis towards isolates of races 1 and 4 ofFusarium oxysporum f.sp.cubense, the Panama wilt pathogen of banana. The seedlings ofMusa balbisiana seedlings treated withP. fluorescens showed less severe wilting and internal discolouration due toF. oxysporum f.sp.cubense infection in greenhouse experiments. In addition to suppressing Panama wilt, bacterized seedlings ofM. balbisiana also showed better root growth and enhanced plant height.     

Transmission of Iris yellow spot virus by Frankliniella fusca and Thrips tabaci (Thysanoptera: Thripidae)

Thrips-transmitted Iris yellow spot virus (IYSV) (Family Bunyaviridae, Genus Tospovirus) affects onion production in the United States and worldwide. The presence of IYSV in Georgia was confirmed in 2003. Two important thrips species that transmit tospoviruses, the onion thrips (Thrips tabaci (Lindeman)) and the tobacco thrips (Frankliniella fusca (Hinds)) are known to infest onion in Georgia. However, T. tabaci is the only confirmed vector of IYSV. Experiments were conducted to test the vector status of F. fusca in comparison with T. tabaci. F. fusca and T. tabaci larvae and adults reared on IYSV-infected hosts were tested with antiserum specific to the nonstructural protein of IYSV through an antigen coated plate ELISA. The detection rates for F. fusca larvae and adults were 4.5 and 5.1%, respectively, and for T. tabaci larvae and adults they were 20.0 and 24.0%, respectively, indicating that both F. fusca and T. tabaci can transmit IYSV. Further, transmission efficiencies of F. fusca and T. tabaci were evaluated by using an indicator host, lisianthus (Eustoma russellianum (Salisbury)). Both F. fusca and T. tabaci transmitted IYSV at 18.3 and 76.6%, respectively. Results confirmed that F. fusca also can transmit IYSV but at a lower efficiency than T. tabaci. To attest if low vector competency of our laboratory-reared F. fusca population affected its IYSV transmission capability, a Tomato spotted wilt virus (Family Bunyaviridae, Genus Tospovirus) transmission experiment was conducted. F. fusca transmitted Tomato spotted wilt virus at a competent rate (90%) suggesting that the transmission efficiency of a competent thrips vector can widely vary between two closely related viruses.     

Insights on genomic diversity of Vibrio spp. through Pan-genome analysis

Purpose

The aquaculture sector is a major contributor to the economic and nutritional security for a number of countries. India’s total seafood exports for the year 2017–2018 accounted for US$ Million 7082. One of the major setbacks in this sector is the frequent outbreaks of diseases often due to bacterial pathogens. Vibriosis is one of the major diseases caused by bacteria of Vibrio spp., causing significant economic loss to the aquaculture sector. The objective of this study was to understand the genetic composition of Vibrio spp.

Methods

Thirty-five complete genomes were downloaded from GenBank comprising seven vibrio species, namely, Vibrio alginolyticus, V. anguillarum, V. campbellii, V. harveyi, V. furnissii, V. parahaemolyticus, and V. vulnificus. Pan-genome analysis was carried out with coding sequences (CDS) generated from all the Vibrio genomes. In addition, genomes were mined for genes coding for toxin-antitoxin systems, antibiotic resistance, genomic islands, and virulence factors.

Results

Results revealed an open pan-genome comprising of 2004 core, 8249 accessory, and 6780 unique genes. Downstream analysis of genomes and the identified unique genes resulted in 312 antibiotic resistance genes, 430 genes coding for toxin and antitoxin systems along with 4802, and 4825 putative virulent genes from genomic island regions and unique gene sets, respectively.

Conclusion

Pan-genome and other downstream analytical procedures followed in this study have the potential to predict strain-specific genes and their association with habitat and pathogenicity.

     

Gene expression enhancement mediated by the 5′ UTR intron of the rice <Emphasis Type="Italic">rubi3</Emphasis> gene varied remarkably among tissues in transgenic rice plants

Introns are important sequence elements that modulate the expression of genes. Using the GUS reporter gene driven by the promoter of the rice (Oryza sativa L.) polyubiquitin rubi3 gene, we investigated the effects of the 5' UTR intron of the rubi3 gene and the 5' terminal 27 bp of the rubi3 coding sequence on gene expression in stably transformed rice plants. While the intron enhanced GUS gene expression, the 27-bp fused to the GUS coding sequence further augmented GUS expression level, with both varying among different tissues. The intron elevated GUS gene expression mainly at mRNA accumulation level, but also stimulated enhancement at translational level. The enhancement on mRNA accumulation, as determined by realtime quantitative RT-PCR, varied remarkably with tissue type. The augmentation by the intron at translational level also differed by tissue type, but to a lesser extent. On the other hand, the 27-bp fusion further boosted GUS protein yield without affecting mRNA accumulation level, indicating stimulation at translation level, which was also affected by tissue type. The research revealed substantial variation in the magnitudes of intron-mediated enhancement of gene expression (IME) among tissues in rice plants and the importance of using transgenic plants for IME studies.     

Process design and optimization of bioethanol production from cassava bagasse using statistical design and genetic algorithm

Abstract

Bioethanol production from agro-industrial residues is gaining attention because of the limited production of starch grains and sugarcane, and food–fuel conflict. The aim of the present study is to maximize the bioethanol production using cassava bagasse as a feedstock. Enzymatic liquefaction, by α-amylase, followed by simultaneous saccharification and fermentation (SSF), using glucoamylase and Zymomonas mobilis MTCC 2427, was investigated for bioethanol production from cassava bagasse. The factors influencing ethanol production process were identified and screened for significant factors using Plackett–Burman design. The significant factors (cassava bagasse concentration (10–50?g/L), concentration of α-amylase (5–25% (v/v), and temperature of fermentation (27–37?°C)) were optimized by employing Box–Behnken design and genetic algorithm. The maximum ethanol concentrations of 25.594?g/L and 25.910?g/L were obtained from Box–Behnken design and genetic algorithm, respectively, under optimum conditions. Thus, the study provides valuable insights in utilizing the cost-effective industrial residue, cassava bagasse, for the bioethanol production.     

Evaluation of the potential of cassava-based residues for biofuels production

Cassava is the third significant source of calories after rice and maize in tropical countries. The annual production of cassava crop is approximately 550 million metric tons (MMT) which generates about 350 MMT of cassava solid residues, including peel, bagasse, stem, rhizome, and leaves. Cassava peel, bagasse, stem, and rhizome can be exploited for solid, liquid and gaseous biofuels production. Biofuels production from cassava starch started in the 1970s and researchers are now extensively studying cassava residues like peel, bagasse, stem, rhizome, and leaves to unravel their applications in biofuels production. However, there are technical and economic challenges to overcome the problems existing in the production of biofuels from cassava-based residues. This review provides a comprehensive summary of the techniques used for biofuels production from various cassava-based residues.