Reduction of fibrillar strain-rate sensitivity in steroid-induced osteoporosis linked to changes in mineralized fibrillar nanostructure.

As bone is used in a dynamic mechanical environment, understanding the structural origins of its time-dependent mechanical behaviour - and the alterations in metabolic bone disease - is of interest. However, at the scale of the mineralized fibrillar matrix (nanometre-level), the nature of the strain-rate dependent mechanics is incompletely understood. Here, we investigate the fibrillar- and mineral-deformation behaviour in a murine model of Cushing's syndrome, used to understand steroid induced osteoporosis, using synchrotron small- and wide-angle scattering/diffraction combined with in situ tensile testing at three strain rates ranging from 10-4 to 10-1 s-1. We find that the effective fibril- and mineral-modulus and fibrillar-reorientation show no significant increase with strain-rate in osteoporotic bone, but increase significantly in normal (wild-type) bone. By applying a fibril-lamellar two-level structural model of bone matrix deformation to fit the results, we obtain indications that altered collagen-mineral interactions at the nanoscale - along with altered fibrillar orientation distributions - may be the underlying reason for this altered strain-rate sensitivity. Our results suggest that an altered strain-rate sensitivity of the bone matrix in osteoporosis may be one of the contributing factors to reduced mechanical competence in such metabolic bone disorders, and that increasing this sensitivity may improve biomechanical performance.

Bone matrix development in steroid-induced osteoporosis is associated with a consistently reduced fibrillar stiffness linked to altered bone mineral quality.

Glucocorticoid-induced osteoporosis (GIOP) is a major secondary form of osteoporosis, with the fracture risk significantly elevated - at similar levels of bone mineral density - in patients taking glucocorticoids compared with non-users. The adverse bone structural changes at multiple hierarchical levels in GIOP, and their mechanistic consequences leading to reduced load-bearing capacity, are not clearly understood. Here we combine experimental X-ray nanoscale mechanical imaging with analytical modelling of the bone matrix mechanics to determine mechanisms causing bone material quality deterioration during development of GIOP. In situ synchrotron small-angle X-ray diffraction combined with tensile testing was used to measure nanoscale deformation mechanisms in a murine model of GIOP, due to a corticotrophin-releasing hormone promoter mutation, at multiple ages (8-, 12-, 24- and 36 weeks), complemented by quantitative micro-computed tomography and backscattered electron imaging to determine mineral concentrations. We develop a two-level hierarchical model of the bone matrix (mineralized fibril and lamella) to predict fibrillar mechanical response as a function of architectural parameters of the mineralized matrix. The fibrillar elastic modulus of GIOP-bone is lower than healthy bone throughout development, and nearly constant in time, in contrast to the progressively increasing stiffness in healthy bone. The lower mineral platelet aspect ratio value for GIOP compared to healthy bone in the multiscale model can explain the fibrillar deformation. Consistent with this result, independent measurement of mineral platelet lengths from wide-angle X-ray diffraction finds a shorter mineral platelet length in GIOP. Our results show how lowered mineralization combined with altered mineral nanostructure in GIOP leads to lowered mechanical competence. SIGNIFICANCE STATEMENT: Increased fragility in musculoskeletal disorders like osteoporosis are believed to arise due to alterations in bone structure at multiple length-scales from the organ down to the supramolecular-level, where collagen molecules and elongated mineral nanoparticles form stiff fibrils. However, the nature of these molecular-level alterations are not known. Here we used X-ray scattering to determine both how bone fibrils deform in secondary osteoporosis, as well as how the fibril orientation and mineral nanoparticle structure changes. We found that osteoporotic fibrils become less stiff both because the mineral nanoparticles became shorter and less efficient at transferring load from collagen, and because the fibrils are more randomly oriented. These results will help in the design of new composite musculoskeletal implants for bone repair.

Towards in situ determination of 3D strain and reorientation in the interpenetrating nanofibre networks of cuticle.

Determining the in situ 3D nano- and microscale strain and reorientation fields in hierarchical nanocomposite materials is technically very challenging. Such a determination is important to understand the mechanisms enabling their functional optimization. An example of functional specialization to high dynamic mechanical resistance is the crustacean stomatopod cuticle. Here we develop a new 3D X-ray nanostrain reconstruction method combining analytical modelling of the diffraction signal, fibre-composite theory and in situ deformation, to determine the hitherto unknown nano- and microscale deformation mechanisms in stomatopod tergite cuticle. Stomatopod cuticle at the nanoscale consists of mineralized chitin fibres and calcified protein matrix, which form (at the microscale) plywood (Bouligand) layers with interpenetrating pore-canal fibres. We uncover anisotropic deformation patterns inside Bouligand lamellae, accompanied by load-induced fibre reorientation and pore-canal fibre compression. Lamination theory was used to decouple in-plane fibre reorientation from diffraction intensity changes induced by 3D lamellae tilting. Our method enables separation of deformation dynamics at multiple hierarchical levels, a critical consideration in the cooperative mechanics characteristic of biological and bioinspired materials. The nanostrain reconstruction technique is general, depending only on molecular-level fibre symmetry and can be applied to the in situ dynamics of advanced nanostructured materials with 3D hierarchical design.

Staggered Fibrils and Damageable Interfaces Lead Concurrently and Independently to Hysteretic Energy Absorption and Inhomogeneous Strain Fields in Cyclically Loaded Antler Bone.

The high toughness and work to fracture of hierarchical composites, like antler bone, involve structural mechanisms at the molecular, nano-, and micro scales, which are not completely explored. A key characteristic of the high energy absorption of such materials is the large hysteresis during cyclic loading, but its origin remains unknown. In situ synchrotron X-ray diffraction tests during tensile loading of antler bone showed heterogeneous fibrillar deformation and hysteresis. To explain the origin of these mechanisms from the nanostructure of antler bone, here we develop a class of finite-element fibril models whose predictions are compared to experimental data across a range of potential composite architectures. We demonstrate that the key structural motif enabling a match to experimental data is an axially staggered arrangement of stiff mineralized collagen fibrils coupled with weak, damageable interfibrillar interfaces.

Uncovering three-dimensional gradients in fibrillar orientation in an impact-resistant biological armour.

The complex hierarchical structure in biological and synthetic fibrous nanocomposites entails considerable difficulties in the interpretation of the crystallographic texture from diffraction data. Here, we present a novel reconstruction method to obtain the 3D distribution of fibres in such systems. An analytical expression is derived for the diffraction intensity from fibres, explaining the azimuthal intensity distribution in terms of the angles of the three dimensional fibre orientation distributions. The telson of stomatopod (mantis shrimp) serves as an example of natural biological armour whose high impact resistance property is believed to arise from the hierarchical organization of alpha chitin nanofibrils into fibres and twisted plywood (Bouligand) structures at the sub-micron and micron scale. Synchrotron microfocus scanning X-ray diffraction data on stomatopod telson were used as a test case to map the 3D fibre orientation across the entire tissue section. The method is applicable to a range of biological and biomimetic structures with graded 3D fibre texture at the sub-micron and micron length scales.

Multiscale alterations in bone matrix quality increased fragility in steroid induced osteoporosis.

A serious adverse clinical effect of glucocorticoid steroid treatment is secondary osteoporosis, enhancing fracture risk in bone. This rapid increase in bone fracture risk is largely independent of bone loss (quantity), and must therefore arise from degradation of the quality of the bone matrix at the micro- and nanoscale. However, we lack an understanding of both the specific alterations in bone quality n steroid-induced osteoporosis as well as the mechanistic effects of these changes. Here we demonstrate alterations in the nanostructural parameters of the mineralized fibrillar collagen matrix, which affect bone quality, and develop a model linking these to increased fracture risk in glucocorticoid induced osteoporosis. Using a mouse model with an N-ethyl-N-nitrosourea (ENU)-induced corticotrophin releasing hormone promoter mutation (Crh(-120/+)) that developed hypercorticosteronaemia and osteoporosis, we utilized in situ mechanical testing with small angle X-ray diffraction, synchrotron micro-computed tomography and quantitative backscattered electron imaging to link altered nano- and microscale deformation mechanisms in the bone matrix to abnormal macroscopic mechanics. We measure the deformation of the mineralized collagen fibrils, and the nano-mechanical parameters including effective fibril modulus and fibril to tissue strain ratio. A significant reduction (51%) of fibril modulus was found in Crh(-120/+) mice. We also find a much larger fibril strain/tissue strain ratio in Crh(-120/+) mice (~1.5) compared to the wild-type mice (~0.5), indicative of a lowered mechanical competence at the nanoscale. Synchrotron microCT show a disruption of intracortical architecture, possibly linked to osteocytic osteolysis. These findings provide a clear quantitative demonstration of how bone quality changes increase macroscopic fragility in secondary osteoporosis.

Mapping QTLs for opaque2 modifiers influencing the tryptophan content in quality protein maize using genomic and candidate gene-based SSRs of lysine and tryptophan metabolic pathway.

KEY MESSAGE: The mapping analysis resulted in identification of five significant QTLs for opaque2 modifiers influencing the tryptophan content in quality protein maize using functional and genomic SSR markers. Quality protein maize (QPM) was developed by selecting genetic modifiers that convert opaque2 mutant containing high lysine and tryptophan. There are several unlinked opaque2 modifier loci (Opm) in QPM whose location, nature and mode of action are not clear. To identify these Opm QTLs, we developed a population of 218 F2:3 individuals from a cross between VQL2 and VQL8, two isogenic QPM inbreds significantly differing in tryptophan content. Based on the data of the F2:3 population, five significant QTLs on chromosomes 5, 7 and 9 with LOD values more than 2.5 were identified and together explained 38.6 % of the total phenotypic variance (R (2)). The Wx1 gene which has influence on the amino acid composition of the maize endosperm was mapped on chromosome 9 near the marker phi022 and also validated by bulk analysis. The QTL near the SSR marker ZmASK3, developed from the aspartate kinase 2 gene of the lysine pathway, mapped on chromosome 5 and had LOD of 2.7 with R (2) of 5.1 %. On chromosome 9, the QTL between the loci umc1430 and bnlg1401 had an LOD of 4.5 with R (2) of 9.1 %, whereas the QTL between the loci bnlg1401 and phi022 had an LOD of 4.2 with R (2) of 8.4 %. The third QTL was observed to be close to the marker umc2207 with an LOD of 4.8 and R (2) of 8.4 %. The identified QTLs will be very useful in the marker-assisted back-cross breeding and transgressive breeding for the development of QPM maize.

Intrafibrillar plasticity through mineral/collagen sliding is the dominant mechanism for the extreme toughness of antler bone.

The inelastic deformability of the mineralised matrix in bones is critical to their high toughness, but the nanoscale mechanisms are incompletely understood. Antler is a tough bone type, with a nanostructure composed of mineralised collagen fibrils ∼100nm diameter. We track the fibrillar deformation of antler tissue during cyclic loading using in situ synchrotron small-angle X-ray diffraction (SAXD), finding that residual strain remains in the fibrils after the load was removed. During repeated unloading/reloading cycles, the fibril strain shows minimal hysteresis when plotted as a function of tissue strain, indicating that permanent plastic strain accumulates inside the fibril. We model the tensile response of the mineralised collagen fibril by a two - level staggered model - including both elastic - and inelastic regimes - with debonding between mineral and collagen within fibrils triggering macroscopic inelasticity. In the model, the subsequent frictional sliding at intrafibrillar mineral/collagen interfaces accounts for subsequent inelastic deformation of the tissue in tension. The model is compared to experimental measurements of fibrillar and mineral platelet strain during tensile deformation, measured by in situ synchrotron SAXD and wide-angle X-ray diffraction (WAXD) respectively, as well as macroscopic tissue stress and strain. By fitting the model predictions to experimentally observed parameters like the yield point, elastic modulus and post-yield slope, extremely good agreement is found between the model and experimental data at both the macro- and at the nanoscale. Our results provide strong evidence that intrafibrillar sliding between mineral and collagen leads to permanent plastic strain at both the fibril and the tissue level, and that the energy thus dissipated is a significant factor behind the high toughness of antler bone.

Symmetrically reduced stiffness and increased extensibility in compression and tension at the mineralized fibrillar level in rachitic bone.

In metabolic bone diseases, the alterations in fibrillar level bone-material quality affecting macroscopic mechanical competence are not well-understood quantitatively. Here, we quantify the fibrillar level deformation in cantilever bending in a mouse model for hereditary rickets (Hpr). Microfocus in-situ synchrotron small-angle X-ray scattering (SAXS) combined with cantilever bending was used to resolve nanoscale fibril strain in tensile- and compressive tissue regions separately, with quantitative backscattered scanning electron microscopy used to measure microscale mineralization. Tissue-level flexural moduli for Hpr mice were significantly (p<0.01) smaller compared to wild-type (~5 to 10-fold reduction). At the fibrillar level, the fibril moduli within the tensile and compressive zones were significantly (p<0.05) lower by ~3- to 5-fold in Hpr mice compared to wild-type mice. Hpr mice have a lower mineral content (24.2±2.1Cawt.% versus 27.4±3.3Ca wt.%) and its distribution was more heterogeneous compared to wild-type animals. However, the average effective fibril modulus did not differ significantly (p>0.05) over ages (4, 7 and 10weeks) between tensile and compressive zones. Our results indicate that incompletely mineralized fibrils in Hpr mice have greater deformability and lower moduli in both compression and tension, and those compressive and tensile zones have similar moduli at the fibrillar level.

Hypophosphatemic rickets is associated with disruption of mineral orientation at the nanoscale in the flat scapula bones of rachitic mice with development.

Metabolic bone disorders such as rickets are associated with altered in vivo muscular force distributions on the skeletal system. During development, these altered forces can potentially affect the spatial and temporal dynamics of mineralised tissue formation, but the exact mechanisms are not known. Here we have used a murine model of hypophosphatemic rickets (Hpr) to study the development of the mineralised nanostructure in the intramembranously ossifying scapulae (shoulder bone). Using position-resolved scanning small angle X-ray scattering (SAXS), we quantified the degree and direction of mineral nanocrystallite alignment over the width of the scapulae, from the load bearing lateral border (LB) regions to the intermediate infraspinous fossa (IF) tissue. These measurements revealed a significant (p<0.05) increase in mineral nanocrystallite alignment in the LB when compared to the IF region, with increased tissue maturation in wild-type mice; this was absent in mice with rickets. The crystallites were more closely aligned to the macroscopic bone boundary in the LB when compared to the IF region in both wild type and Hpr mice, but the degree of alignment was reduced in Hpr mice. These findings are consistent with a correlation between the nanocrystallites within fibrils and in vivo muscular forces. Thus our results indicate a relevant mechanism for the observed increased macroscopic deformability in rickets, via a significant alteration in the mineral particle alignment, which is mediated by an altered spatial distribution of muscle forces.

In situ multi-level analysis of viscoelastic deformation mechanisms in tendon collagen.

Tendon is a hydrated multi-level fibre composite, in which time-dependent behaviour is well established. Studies indicate significant stress relaxation, considered important for optimising tissue stiffness. However, whilst this behaviour is well documented, the mechanisms associated with the response are largely unknown. This study investigates the sub-structural mechanisms occurring during stress relaxation at both the macro (fibre) and nano (fibril) levels of the tendon hierarchy. Stress relaxation followed a two-stage exponential behaviour, during which structural changes were visible at the fibre and fibril levels. Fibril relaxation and fibre sliding showed a double exponential response, while fibre sliding was clearly the largest contributor to relaxation. The amount of stress relaxation and sub-structural reorganisation increased with increasing load increments, but fibre sliding was consistently the largest contributor to stress relaxation. A simple model of tendon viscoelasticity at the fibril and fibre levels has been developed, capturing this behaviour by serially coupling a Voigt element (collagen fibril), with two Maxwell elements (non-collagenous matrix between fibrils and fibres). This multi-level analysis provides a first step towards understanding how sub-structural interactions contribute to viscoelastic behaviour. It indicates that nano- and micro-scale shearing are significant dissipative mechanisms, and the kinetics of relaxation follows a two-stage exponential decay, well fitted by serially coupled viscoelastic elements.

Growth promotion of wheat seedlings by Exiguobacterium acetylicum 1P (MTCC 8707) a cold tolerant bacterial strain from the Uttarakhand Himalayas.

Exiguobacterium acetylicum strain 1P (MTCC 8707) is a gram-positive, rod-shaped, yellow pigmented bacterium isolated from soil on nutrient agar plates at 4°C. The identity of the bacterium was arrived on the basis of the biochemical characterization, BIOLOG sugar utilization pattern and sequencing of the 16S rRNA gene. It grew at temperatures ranging from 4 to 42°C, with temperature optima at 30°C. It expressed multiple plant growth promotion attributes such as phosphate solubilization, indole acetic acid (IAA), siderophore and hydrogen cyanide (HCN) production, differentially at suboptimal growth temperatures (15 and 4°C). At 15°C it solubilized phosphate (21.1 µg of P ml(-1) day(-1)), and produced IAA (14.9 µg ml(-1) day(-1)) in tryptophan amended media. Qualitative detection of siderophore production and HCN were possible at 15°C. At 4°C it retained all the plant growth promotion attributes. Seed bacterization with the isolate, positively influenced the growth and nutrient uptake parameters of wheat seedlings in glass house studies at suboptimal cold growing temperatures.

Variability of nutritional and cooking quality in bean (Phaseolus vulgaris L) as a function of genotype.

Screening of natural biodiversity for the better quality traits are of prime importance for quality breeding programs. The objective of this investigation was to select candidate accession of bean having high concentrations of protein as well as macro and micro minerals with good cooking quality for use as parents in breeding programme for these compounds. Thirty-five accessions of bean (Phaseolus vulgaris L) were field grown and their seeds were analyzed for their cooking quality and nutritional composition. Wide variations were observed in most of the measurements e.g. protein (18.7-26.2%), iron (79.4-137.6 ppm) and hardness after cooking (4.65-9.88 Kg) suggesting that there are considerable levels of genetic diversity. Across all accessions the concentration of potassium was negatively correlated with protein (r = -0.43, P < 0.05). Concentrations of protein was significantly greater in accessions VIII, XIII and XIX compared to other accessions analyzed. Iron concentrations were greatest (137 ppm) in XIX and lowest (79 ppm) in XXVII. Lines with less cooking time were line III, X, XXVI, XXX and XXXI. Bean line XIX contains high protein (24.9%) with high zinc (33.3 ppm) and highest iron (137.6 ppm), but it has high hardness after cooking (7.32 kg). Four clusters were computed by cluster analysis that explained quite a good variation in the traits. The great variability for these attributes suggests that these selected accessions may be useful as parents in hybridization programs to produce bean with value-added traits. This information was also potentially useful for pulse breeders working on the development of new varieties.

Fracture of bone tissue: The 'hows' and the 'whys'.

The mechanical performance of bone is of paramount importance for the quality of life we experience. The structural integrity of bone, its hierarchical structure, organisation and its physicochemical constitution, all influence its ability to withstand loads, such as those seen occasionally in everyday life loading scenarios, which are either above the norm, prolonged, or repetitive. The present review explores three interconnected areas of research where significant progress has been made lately: (i) The recorded mechanical behaviour of bone and the way it fails; (ii) the inner architecture, organisational, hierarchical structure of bone tissue; and (iii) the bone properties at the micro/nanostructural and biophysical level. Exercising a line of thought along a structure/function based argument we advance from 'how' bone fractures to 'why' it fractures, and we seek to obtain a fresh insight in this field.

Relative changes in phosphatase activities as influenced by source and application rate of organic composts in field crops.

Potential impact of different levels and sources of organic composts on activities of phosphatases (acid and alkaline phosphatase, phosphodiesterase, and inorganic pyrophosphatase) was studied after three years of continuous application. Enzyme activities were compared with microbial biomass P and available P. Experimental plots were divided based on the organic source into three groups: those receiving farmyard manure (FYM), vermicompost (VC) and Lantana compost (LC). Microbial biomass P (11.7 g kg(-1) soil), available P (24.0 g kg(-1) soil) and acid phosphatase (1.3 mg g(-1) p-NP g(-1) soil h(-1)) was highest in highest dose of VC. Acid phosphatase activity was high in all plots, including those where microbial biomass P levels were low. Most of the phosphatase activities were significantly correlated with available P in FYM and VC. These relationships were negative for LC treatments. Results showed that application of earthworm casts is helpful in faster transformation of organic P by facilitating better environment to microbes and plant roots.

Cold tolerance and plant growth promotion potential of Serratia marcescens strain SRM (MTCC 8708) isolated from flowers of summer squash (Cucurbita pepo).

AIM: To determine the cold tolerance and plant growth promotion potential of Serratia marcescens strain SRM (MTCC 8708). METHODS AND RESULTS: Serratia marcescens strain SRM was isolated from the flowers of summer squash plants, showing no apparent symptoms of yellow vine disease. It was evaluated for growth and plant growth promotion attributes at 15 and 4 degrees C. At 15 degrees C, the isolate was able to solubilize 76.6 microg ml(-1) of P and produce Indole Acetic Acid, IAA (11.1 microg ml(-1)). HCN and siderophore production were also detected at 15 degrees C. The isolate retained all the plant growth promotion traits at 4 degrees C. Seed bacterization with the isolate significantly enhanced plant biomass and nutrient uptake of wheat seedlings grown in cold temperatures. CONCLUSION: Serratia marcescens strain SRM is a promising cold-tolerant isolate that can significantly influence wheat seedling growth at cold temperatures. SIGNIFICANCE AND IMPACT OF THE STUDY: This strain can be employed as a bioinoculant in cold temperature conditions.

Raman imaging of two orthogonal planes within cortical bone.

The lamellar bone's strength is mainly affected by the organization of its mineralized collagen fibers and material composition. In the present study, Raman microspectroscopic and imaging analyses were employed to study a normal human femoral midshaft bone cube-like specimen with a spatial resolution of approximately 1-2 microm. Identical bone lamellae in both longitudinal and transverse directions were analyzed, which allowed us to separate out orientation and composition dependent Raman lines, depending on the polarization directions. This approach gives information about lamellar bone orientation and variation in bone composition. It is shown that the nu1 PO4 to amide I ratio mainly displays lamellar bone orientation; and nu2 PO4 to amide III and CO3 to nu2 PO4 ratios display variation in bone composition. The nu2 PO4 to amide III ratio is higher in the interstitial bone region, whereas the CO3 to nu2 PO4 ratio has lower values in the same region. The present study provides fresh insights into the organization of a lamellar bone tissue from two orthogonal orientations.

Mapping QTLs for popping ability in a popcorn x flint corn cross.

Popping expansion volume (PEV) in popcorn (Zea mays L.) is a distinct heritable character and defined as the ratio of the volume after popping to the volume before popping. PEV is quantitatively inherited and 3-4 genes/quantitative trait loci (QTLs) have been implicated. In the present study, we have dissected the quantitative PEV into two component traits, viz., flake volume (FV) and percent unpopped kernels (UPK), and mapped QTLs using SSR markers for all three traits with 194 F3 families derived from a popcorn (A-1-6) x flint corn (V273) cross. Heritability (broad sense) estimates for PEV, FV and UPK based on F3 mean bases were 0.72, 0.54 and 0.68, respectively. The QTL analyses for the three traits based on combined environment data were performed by composite interval mapping using QTL cartographer. Four QTLs were identified for PEV on chromosomes 1, 3, 8 and 10, which together explained 62% of the phenotypic variance (sigma2p). Four QTLs were found on chromosomes 1, 5, 9 and 10 for FV (explaining 44% of sigma2p) and five QTLs for UPK on chromosomes 1, 3, 4, 5 and 9 (explaining 57% of sigma2p). The relative efficiency estimates of marker-based selection in comparison to phenotypic selection for PEV (1.10), FV (1.22) and UPK (1.11) indicated that marker-based selection could be relatively more efficient. The QTL on chromosome 1S for PEV was found to be most significant, where QTLs for hard endosperm starch concentration had been detected earlier.

Spiral twisting of fiber orientation inside bone lamellae.

The secondary osteon - a fundamental building block in compact bone - is a multilayered cylindrical structure of mineralized collagen fibrils arranged around a blood vessel. Functionally, the osteon must be adapted to the in vivo mechanical stresses in bone at the level of its microstructure. However, questions remain about the precise mechanism by which this is achieved. By application of scanning x-ray diffraction with a micron-sized synchrotron beam, along with measurements of local mineral crystallographic axis direction, we reconstruct the three-dimensional orientation of the mineralized fibrils within a single osteon lamella ( approximately 5 mum). We find that the mineralized collagen fibrils spiral around the central axis with varying degrees of tilt, which would - structurally - impart high extensibility to the osteon. As a consequence, strains inside the osteon would have to be taken up by means of shear between the fibrils.

Two-generation marker-aided backcrossing for rapid conversion of normal maize lines to quality protein maize (QPM).

The low nutritive value of maize endosperm protein is genetically corrected in quality protein maize (QPM), which contains the opaque 2 gene along with numerous modifiers for kernel hardness. We report here a two generation marker-based backcross breeding program for incorporation of the opaque 2 gene along with phenotypic selection for kernel modification in the background of an early maturing normal maize inbred line, V25. Using the flanking marker distances from opaque 2 gene in the cross V 25 xCML 176, optimum population size for the BC(2) generation was computed in such a way that at least one double recombinant could be obtained. Whole genome background selection in the BC(2) generation identified three plants with 93 to 96% recurrent parent genome content. The three BC(2)F(2) families derived from marker identified BC(2) individuals were subjected to foreground selection and phenotypic selection for kernel modification. The tryptophan concentration in endosperm protein was significantly enhanced in all the three classes of kernel modification viz., less than 25%, 25--50% and more than 50% opaqueness. BC(2)F(3) lines developed from the hard endosperm kernels were evaluated for desirable agronomic and biochemical traits in replicated trials and the best line was chosen to represent the QPM version of V25, with tryptophan concentration of 0.85% in protein. The integrated breeding strategy reported here can be applied to reduce genetic drag as well as the time involved in a conventional line conversion program, and would prove valuable in rapid development of specialty corn germ plasm.