QTL analysis for grain quality traits in 2 BC2F2 populations derived from crosses between Oryza sativa cv Swarna and 2 accessions of O. nivara.

The appearance and cooking quality of rice determine its acceptability and price to a large extent. Quantitative trait loci (QTLs) for 12 grain quality traits were mapped in 2 mapping populations derived from Oryza sativa cv Swarna × O. nivara. The BC(2)F(2) population of the cross Swarna × O. nivara IRGC81848 (population 1) was evaluated during 2005 and that from Swarna × O. nivara IRGC81832 (population 2) was evaluated during 2006. Linkage maps were constructed using 100 simple sequence repeat (SSR) markers in population 1 and 75 SSR markers in population 2. In all, 21 QTLs were identified in population 1 (43% from O. nivara) and 37 in population 2 (38% QTLs from O. nivara). The location of O. nivara-derived QTLs mp1.2 for milling percent, kw6.1 for kernel width, and klac12.1 for kernel length after cooking coincided in the 2 populations and appear to be useful for Marker Assisted Selection (MAS). Four QTLs for milling percent, 1 QTL each for amylose content, water uptake, elongation ratio, 2 QTLs for kernel width, and 3 QTLs for gel consistency, each explained more than 20% phenotypic variance. Three QTL clusters for grain quality traits were close to the genes/QTLs for shattering and seed dormancy. QTLs for 4 quality traits were associated with 5 of the 7 major yield QTLs reported in the same 2 mapping populations. Useful introgression lines have been developed for several agronomic traits. It emerges that 40% O. nivara alleles were trait enhancing in both populations, and QTLs for grain quality overlapped with yield meta-QTLs and QTLs for dormancy and seed shattering.

ISSR markers based on GA and AG repeats reveal genetic relationship among rice varieties tolerant to drought, flood, or salinity.

Drought, flood, salinity, or a combination of these limits rice production. Several rice varieties are well known for their tolerance to specific abiotic stresses. We determined genetic relationship among 12 rice varieties including 9 tolerant to drought, flood, or salinity using inter-simple sequence repeat (ISSR) markers. Based on all markers, the nine tolerant varieties formed one cluster distinct from the cluster of three control varieties. The salt-tolerant varieties were closest to two flood-tolerant varieties, and together they were distinct from the drought-tolerant varieties. (GA)(8)YG was the most informative primer, showing the highest polymorphic information content (PIC) and resolving power (Rp). The drought-, flood-, and salt-tolerant varieties grouped in three distinct clusters within the group of tolerant varieties, when (GA)(8)YG was used. Sabita was the only exception. The two aus varieties, Nagina22 and FR13A, were separated and grouped with the drought- and flood-tolerant varieties, respectively, but they were together in dendrograms based on other primers. The results show that ISSR markers associated with (GA)(8)YG delineated the three groups of stress-tolerant varieties from each other and can be used to identify genes/new alleles associated with the three abiotic stresses in rice germplasm.

Cell mimetic lateral stabilization of outer cell mimetic bilayer on polymer surfaces by peptide bonding and their blood compatibility.

The biological lipid bilayer membranes are stabilized laterally with the help of integral proteins. We have simulated this with an optimized ternary phospholipid/glycolipid/cholesterol system, and stabilized laterally on functionalized poly methyl methacrylate (PMMA) surfaces, using albumin, heparin, and polyethylene glycol as anchors. We have earlier demonstrated the differences due to orientation and packing of the ternary phospholipid monolayers in relation to blood compatibility (Kaladhar and Sharma, Langmuir 2004;20:11115-11122). The structure of albumin is changed here to expose its interior hydrophobic core by treating with organic solvent. The interaction between the hydrophobic core of the albumin molecule and the hydrophobic core of the lipid molecules is confirmed by incorporating the molecule into bilayer membranes. The secondary structure of the membrane incorporated albumin is studied by CD spectral analysis. The structure of the altered albumin molecule contains more beta-sheet as compared to the native albumin. This conformation is also retained in membranes. The partitioning of the different anchors based on its polarity and ionic interactions in the monolayer is studied from the pressure-area (pi-A) isotherm of the lipid monolayers at the air/water interface using Langmuir-Blodgett (LB) trough facility. Such two monolayers are deposited onto the functionalized PMMA surface using LB trough and crosslinked by carbodiimide chemistry. The structure of the deposited bilayer is studied by depth analysis using contact mode AFM in dry conditions. The stabilized bilayer shows stability up to 1 month by contact angle studies. Preliminary blood compatibility studies reveal that the calcification, protein adsorption, as well as blood-cell adhesion is significantly reduced after the surface modification. The reduced adsorption of ions, proteins, and cells to the modified surfaces may be due to the fluidity of the microenvironment along with the contribution of the mobile PEG groups at the surface and the phosphorylcholine groups of the phospholipids. The stability of the anchored bilayer under low shear stress conditions promises that the laterally stabilized supported bilayer system can be used for low shear applications like small diameter vascular graft and modification of biosensors, and so forth.

Cell mimetic monolayer supported chitosan-haemocompatibility studies.

Chitosan is a natural polymer, widely explored for biomedical and tissue engineering applications. However the thrombogenic nature limits their application in blood contacting devices and implants. Here, we have attempted to understand the haemocompatibility of chitosan by immobilizing a monolayer of cell mimetic lipid compositions. The phosphatidylcholine/cholesterol/galactocerebroside lipid composition (PC/Chol/GalC, 1:0.35:0.125) was deposited onto the chitosan films. Characterization of the modified surface was done by sessile drop contact angle measurement. The contact angle of the chitosan film reduced from 80.65 +/- 1.4 to 23.5 +/- 1.9 after the surface modification. Swelling nature of chitosan seemed to influence the orientation and packing of the lipid monolayer. In vitro calcification studies with metastable salt solution indicated increased calcification on the modified surface. This may be due to formation of nuclei for calcification on the expanding monolayer. The preliminary haemocompatibility studies with washed platelets, leukocytes and erythrocytes showed overall reduction in blood cell adhesion to the modified surfaces. Scanning electron microscopy was used for morphological characterization of platelet adhesion and activation on the surfaces. On the bare chitosan surface, fully spread platelets with extending pseudopodia indicated platelet activation. The smooth surface of the modified film did not activate platelets. These studies showed that, though the lipid monolayer on chitosan film is able to reduce the over all blood cell adhesion and platelet activation it is prone to calcification.

Nano-anisotropic surface coating based on drug immobilized pendant polymer to suppress macrophage adhesion response.

Exploring drug molecules for material design, to harness concepts of nano-anisotropy and ligand-receptor interactions, are rather elusive. The aim of this study is to demonstrate the bottom-up design of a single-step and bio-interactive polymeric surface coating, based on drug based pendant polymer. This can be applied on to polystyrene (PS) substrates, to suppress macrophage adhesion and spreading. The drug molecule is used in this coating for two purposes. The first one is drug as a "pendant" group, to produce nano-anisotropic properties that can enable adhesion of the coatings to the substrate. The second purpose is to use the drug as a "ligand", to produce ligand-receptor interaction, between the bound ligand and receptors of albumin, to develop a self-albumin coat over the surface, by the preferential binding of albumin in biological environment, to reduce macrophage adhesion. Our in silico studies show that, diclofenac (DIC) is an ideal drug based "ligand" for albumin. This can also act as a "pendant" group with planar aryl groups. The combination of these two factors can help to harness, both nano-anisotropic properties and biological functions to the polymeric coating. Further, the drug, diclofenac (DIC) is immobilized to the polyvinyl alcohol (PVA), to develop the pendant polymer (PVA-DIC). The interaction of bound DIC with the albumin is a ligand-receptor based interaction, as per the studies by circular dichroism, differential scanning calorimetry, and SDS-PAGE. The non-polar π-π* interactions are regulating; the interactions between PVA bound DIC-DIC interactions, leading to "nano-anisotropic condensation" to form distinct "nano-anisotropic segments" inside the polymeric coating. This is evident from, the thermo-responsiveness and uniform size of nanoparticles, as well as regular roughness in the surface coating, with similar properties as that of nanoparticles. In addition, the hydrophobic DIC-polystyrene (PS) interactions, between the PVA-DIC coating and PS-substrate produce improved coating stability. Subsequently, the PVA-DIC coated substrate has the maximum capacity to suppress the macrophage (RAW 264.7 cell line) adhesion and spreading, which is partly due to wavy-surface topography of hydrophilic PVA and preferential albumin binding capacity of PVA bound DIC. Our result shows that, such surfaces suppress the macrophages, even under stimulation with lipopolysaccharide (LPS). The modified tissue culture plates can be used as an in vitro tool, to study the macrophage response under low spatial cues.

Cell-mimetic coatings for immune spheres.

Extrinsically induced or engineered cells are providing new therapeutic means in emerging fields such as cell therapeutics, immunomodulation and regenerative medicine. We are demonstrating a spatial induction method using lipid coatings, which can change signal presentation strength from material surface to adherent macrophage cells, that induce early cell-cell interaction leading to organotypic morphology. For that, we have developed a cell mimetic lipid coating with a rafts size to the order of transmembrane proteins (<10 nm) with enhanced lateral elastic properties. Such surface coatings are capable of reducing adherent macrophage spreading, while enabling early induction of cell-cell interaction to form organotypic macrophage colonies or "spheres" (M-spheres).

Mapping and introgression of QTL for yield and related traits in two backcross populations derived from Oryza sativa cv. Swarna and two accessions of O. nivara.

Advanced backcross QTL (AB-QTL) analysis was carried out in two Oryza nivara-derived BC2F2 populations. For nine traits, we identified 28 QTL in population 1 and 26 QTL in population 2. The two most significant yield-enhancing QTL, yldp9.1 and yldp2.1 showed an additive effect of 16 and 7 g per plant in population 1, while yld2.1 and yld11.1 showed an additive effect of 11 and 10 g per plant in population 2. At least one O. nivara-derived QTL with a phenotypic variance of >15% was detected for seven traits in population 1 and three traits in population 2. The O. nivara-derived QTL ph1.1, nt12.1, nsp1.1, nfg1.1, bm11.1, yld2.1 and yld11.1 were conserved at the same chromosomal locations in both populations. Two major QTL clusters were detected at the marker intervals RM488-RM431 and RM6-RM535 on chromosomes 1 and 2, respectively. The colocation of O. nivara-derived yield QTL with yield meta-QTL on chromosomes 1, 2 and 9 indicates their accuracy and consistency. The major-effect QTL reported in this study are useful for marker-assisted breeding and are also suitable for further fine mapping and candidate gene identification.

Supported cell mimetic monolayers and their interaction with blood.

Surface modification using supported monolayers of phosphorylcholine containing phospholipids has been an accepted strategy for developing blood-contacting materials. We present a detailed study of the blood compatibility of the supported monolayers of phospholipid, glycolipid, and cholesterol (Chol) binary and ternary lipid combinations using in vitro techniques. The packing and orientation of these monolayers have been correlated with the blood compatibility. We have used phosphatidylcholine (PTC) for phospholipid, galactocerebroside (Gal) for glycolipid, and Chol based on the headgroup structure to represent the major lipid components of the endothelial luminal cell membrane. The interfacial behavior of various combinations of PTC, Gal, and Chol monolayers have been studied at the air/water interface and deposited on hydrophobic polycarbonate (PC) polymer substrates with the help of the Langmuir-Blodgett trough. The packing and orientation of the supported monolayers have been varied by means of changing the lipid composition rather than the deposition parameters. This approach seems to be more similar to the in vivo conditions. The different supported monolayer surfaces prepared accordingly are (1) a closely packed ordered hydrophobic surface, PC modified with the combination PTC/Chol/Gal (1:0.35:0.125), (2) a loosely packed ordered hydrophobic surface, PC modified with the combination PTC/Chol (1:0.35), and (3) a closely packed ordered hydrophilic surface, PC modified with the combination PTC/Chol (1:0.7). An optimized modified surface (PTC/Chol/Gal, 1:0.35:0.125) has been identified on the basis of the maximum transfer ratio from the air/water interface and characterized by using atomic force microscopy. The concentration of Chol has been found to be an important parameter, which influences the transfer ratio. The Gal improves the monolayer integrity under a reduced Chol concentration. The blood compatibility of these supported monolayers was studied by protein adsorption, blood cell adhesion, and calcification. The tightly packed ordered hydrophobic surface (PTC/Chol/Gal, 1:0.35:0.125), has been found to be more blood compatible because of reduced blood cell adhesion and calcification. This surface also promotes albumin adsorption and may be the reason for the reduced platelet activation, while in the case of the loosely packed ordered hydrophobic surface (PTC/Chol, 1:0.35) the protein adsorption also has been reduced along with the blood cell adhesion and calcification. When the ordered hydrophilic surface (PTC/Chol, 1: 0.7) of the monolayer has been exposed, the blood cell adhesions as well as the overall protein adsorption were significantly reduced. However, the packing of the phosphorylcholine moieties of the polar headgroup has been affecting the calcification on the surface. We have observed an increase in calcification to the surface modified with the loosely packed polar headgroup, from a relative study on chitosan and chitosan modified with the monolayer of PTC. These findings are helpful for the surface modifications for blood-contacting materials using this strategy.