Milestones in chloroplast genetic engineering: an environmentally friendly era in biotechnology

Chloroplast genomes defied the laws of Mendelian inheritance at the dawn of plant genetics, and continue to defy the mainstream approach to biotechnology, leading the field in an environmentally friendly direction. Recent success in engineering the chloroplast genome for resistance to herbicides, insects, disease and drought, and for production of biopharmaceuticals, has opened the door to a new era in biotechnology. The successful engineering of tomato chromoplasts for high-level transgene expression in fruits, coupled to hyper-expression of vaccine antigens, and the use of plant-derived antibiotic-free selectable markers, augur well for oral delivery of edible vaccines and biopharmaceuticals that are currently beyond the reach of those who need them most.     

The Constitutive Heat Shock Protein-70 Is Required for Optimal Expression of Myelin Basic Protein During Differentiation of Oligodendrocytes

To further elucidate the role of the constitutive heat shock protein-70 (HSC70) as a chaperone for the synthesis of myelin basic protein (MBP), HSC70 content was decreased in oligodendrocyte precursor cells prior to MBP expression either by transfection with an antisense oligonucleotide specific for HSC70, or by exposure to low levels of quercetin, a bioflavonoid known to decrease synthesis of HSC70. As these cells underwent differentiation in vitro, antisense treatment decreased HSC70 levels to 66% of controls. At the same time, a sharp induction resulted in the stress-inducible heat shock protein-70 (HSP70). Levels of two other stress proteins increased as well, namely, the 25-kDa heat shock protein (HSP25) and the 78-kDa glucose regulated protein (GRP78). MBP synthesis proceeded over a normal time course, but at only 50% of control values. As HSC70 content returned to normal, MBP synthesis was also restored to normal levels. Quercetin reduced the expression of HSC70 to an even greater extent than transfection, and prevented the induction of HSP70. In contrast to antisense-treated cells, MBP synthesis was essentially blocked in quercetin-treated cells even though levels of HSP25 and GRP78 increased. Taken together, these observations (a) indicate that HSP70 partially compensates for decreased chaperoning of nascent MBP by HSC70 (HSC70 and HSP70 are closely related and perform similar functions); (b) preclude the involvement of HSP25 and GRP78 in MBP synthesis; and (c) emphasize the requirement of HSC70 for optimal synthesis of MBP.     

Arbuscular Mycorrhizal Fungi Enhance Zinc and Nickel Uptake from Contaminated Soil by Soybean and Lentil

Generally, soils in Pakistan are deficient in P and N. Due to intensive cropping and irrigation, Pakistani soils have also become deficient in micronutrients such as Zn, Fe, Cu, and Mn. Arbuscular mycorrhizal fungi, which form symbiotic associations with roots of most land plants, are known to enhance uptake of P and trace elements such as Cu, Ni, Pb, and Zn. The present study was conducted to investigate the role of arbuscular mycorrhizae (AM) in uptake of nickel (Ni) and zinc (Zn) by crops viz. soybean (Glycine max (L.) Merrill) and lentil (Lens culinaris Medic). Zn and Ni were applied as ZnSO₄ 7H₂O and NiCl₂ respectively, in four concentrations (0.0, 1.0, 3.0, and 5.0 g kg^-1 soil). AM inoculum consisted of sand containing sporocarps, spores, and AMF infected root pieces from a pot culture of Glomus mosseae. Control plants received pot culture filtrate containing soil microflora minus AM fungal propagules. A significant difference (p < 0.05) was observed in the dry weights of roots and shoots of the mycorrhizal (M) and nonmycorrhizal (NM) cereal plants. The sievate-amended treatments did not stimulate plant growth to the same extent as the AM fungal amended treatments. Trace metals inhibited the extent of mycorrhizal colonization of the cereal roots. The concentrations of the trace metals in the plant tissues of 12-week old cereal plants were found significantly (p < 0.05) higher in M than NM plants. These results indicate that mycorrhize can be used as effective tools to supply sufficient Zn in generally Zn-deficient Pakistani soils and to ameliorate the toxicity of trace metals in polluted soils. The contents of Ni in mycorrhizal soybean plant tissues were higher than those in the mycorrhizal lentil plant tissues. The implications of these results in mycorrhizo remediation of agricultural soils are discussed.     

A High Through-put Procedure for Capturing Microsatellites from Complex Plant Genomes

A method is outlined for large-scale isolation and characterization of microsatellite sequences from complex plant genomes. The method presented here differs from the previously published procedures in the use of randomly sheared (nebulized) genomic DNA for adapter-ligation, rigorous removal of biotinylated oligos, and high-density colony blots for constructing enriched libraries. Using this method we have constructed cotton microsatellite enriched libraries with over 20% (high stringency screening) or 75% (by random sequencing). Thus far we have identified and sequenced over 500 cotton microsatellites using this procedure. The procedure can be used to generate enriched SSR libraries from genomic DNA in about one week. High throughput screening and automated DNA sequencing can be accomplished in less than one month.     

Two nucleotide sugar transporters are important for cell wall integrity and full virulence of Magnaporthe oryzae

Cell wall polysaccharides play key roles in fungal development, virulence, and resistance to the plant immune system, and are synthesized from many nucleotide sugars in the endoplasmic reticulum (ER)-Golgi secretory system. Nucleotide sugar transporters (NSTs) are responsible for transporting cytosolic-derived nucleotide sugars to the ER lumen for processing, but their roles in plant-pathogenic fungi remain to be revealed. Here, we identified two important NSTs, NST1 and NST2, in the rice blast fungus Magnaporthe oryzae. Both NSTs were localized in the ER, which was consistent with a function in transporting nucleotide sugar for processing in the ER. Sugar transport property analysis suggested that NST1 is involved in transportation of mannose and glucose, while NST2 is only responsible for mannose transportation. Accordingly, deletion of NSTs resulted in a significant decrease in corresponding soluble saccharides abundance and defect in sugar utilization. Moreover, both NSTs played important roles in cell wall integrity, were involved in asexual development, and were required for full virulence. The NST mutants exhibited decreasing external glycoproteins and exposure of inner chitin, which resulted in activation of the host defence response. Altogether, our results revealed that two sugar transporters are required for fungal cell wall polysaccharides accumulation and full virulence of M. oryzae.     

Pre-Breeding Genetic Diversity Assessment of Tomato (<i>Solanum lycopersicum</i> L.) Cultivars Based on Molecular,Morphological and Physicochemical Parameters

Appropriate knowledge of the parental cultivars is a pre-requisite for a successful breeding program. This study characterized fruit yield, quality attributes, and molecular variations of ten tomato cultivars during three consecutive generations under greenhouse conditions. Peto 86, Castle Rock, and Red Star cultivars showed the highest fruit yield (kg/plant), total phenolic compounds (TPC), and sap acidity. Principal component analysis categorized the evaluated fruit yield into three groups based on their quality attributes. A robust positive correlation appeared among traits inside each group. A positive correlation was likewise noticed between the first and the second groups. However, a negative correlation was detected between the first, the second and the third group. Molecular profiling, using seven inter-simple sequence repeat (ISSR) primers, produced 60 loci, including 49 polymorphic loci. The molecular analysis also pinpointed the highest genetic similarity (0.92) between P73 and Moneymaker, while the lowest genetic similarity (0.46) was observed between Castle Rock and Moneymaker. The cultivars P73 and Moneymaker showed the lowest genetic distance (2.24), while the highest genetic distance (5.92) was observed between Super Marmand and Peto86, on the one hand, and between Castle Rock and Moneymaker, on the other hand. The chemical analysis of fruit sap indicated the highest levels of TPC, total flavonoids, anthocyanin, ascorbic acid and total soluble solids in Peto 86 and Castle Rock cultivars. Phylogeny analysis of tomato cultivars based on morphological and molecular attributes indicated four distinct clades. Peto 86, Castle Rock, and Red star cultivars can be recommended for the tomato hybridization breeding programs in the future, with other tomato cultivars as potentially high-yielding parents.     

Insecticidal Potential of α-Pinene and β-Caryophyllene against <i>Myzus persicae</i> and Their Impacts on Gene Expression

Myzus persicae (M. persicae) is now considered a threat to agricultural crops due to economic losses. Numerous synthetic insecticides applied every year against M. persicae, are reported to be unsafe for environment, humans, and beneficial insects. Furthermore, several species of Myzus have been found to develop resistance due to over application of these insecticides. Therefore, it is required to find some novel insecticide that would be safe for the environment as well as for humans. In the current study, two major pure constituents α-pinene and β-caryophyllene were evaluated for their insecticidal potential against M. persicae using a fumigant toxicity assay. Furthermore, impact of α-pinene and β-caryophyllene on expression of five different genes, e.g., HSP 60, FPPS I, OSD, TOL and ANT responsible for reproduction, dispersion, and growth of M. persicae has also been investigated. To perform fumigant toxicity assay, five different concentrations (3.5, 4, 4.5, 5 and 6 μL L⁻¹) of α-pinene and β-caryophyllene were prepared. Lethal concentration (LC) was calculated, and gene expression studies were executed through qRT PCR at LC₃₀ of α-pinene and β-caryophyllene. Both constituents demonstrated excellent fumigant toxicity effects against M. persicae at all five concentrations. However, α-pinene shows significantly better results (98%) as compared to β-caryophyllene (80%) after 72 h at 6 μL L⁻¹ of dose. The highest upregulation in expression was demonstrated at LC₃₀ dose of α-pinene in five in three out of five genes understudy (TOL, ANT, and FPPS I). Conversely, two genes HSP 60 and OSD demonstrated downregulation at LC₃₀ dose of β-caryophyllene. Conclusively, our results highlighted the promising insecticidal potential of both compounds α-pinene and β-caryophylleneby interfering with the reproduction and development related processes in M. persicae, allowing us to recommend the phytoconstituents under investigation as an ecofriendly alternative to synthetic insecticides.     

Nutritionally rich biochemical profile in essential oil of various Mentha species and their antimicrobial activities

Protoplasma - The Mentha species of family Lamiaceae are famous for their flavor and are commercially used in many food products worldwide. They are widely used to cure digestive problems as well...