Doubled-haploid production in chickpea (<Emphasis Type="Italic">Cicer arietinum</Emphasis> L.): role of stress treatments

This is the first report on the production of double-haploid chickpea embryos and regenerated plants through anther culture using Canadian cultivar CDC Xena (kabuli) and Australian cultivar Sonali (desi). Maximum anther induction rates were 69% for Sonali and 63% for CDC Xena. Under optimal conditions, embryo formation occurred within 15–20 days of culture initiation with 2.3 embryos produced per anther for CDC Xena and 2.0 embryos per anther for Sonali. For anther induction, the following stress treatments were used: (1) flower clusters were treated at 4°C for 4 days, (2) anthers were subjected to electric shock treatment of three exponentially decaying pulses of 50–400 V with 25 μF capacitance and 25 Ω resistance, (3) anthers were centrifuged at 168–1,509g for 2–15 min, and finally (4) anthers were cultured for 4 days in high-osmotic pressure (563 mmol) liquid medium. Anthers were then transferred to a solid embryo development medium and, 15–20 days later, embryo development was observed concomitant with a small amount of callus growth of 0.1–3 mm. Anther-derived embryos were regenerated on plant regeneration medium. Electroporation treatment of anthers enhanced root formation, which is often a major hurdle in legume regeneration protocols. Cytological studies using DAPI staining showed a wide range of ploidy levels from haploid to tetraploid in 10–30-day-old calli. Flow cytometric analysis of calli, embryos and regenerated plants showed haploid profiles and/or spontaneous doubling of the chromosomes during early regeneration stages.     

Interaction of Vitamin D Receptor with HLA DRB1*0301 in Type 1 Diabetes Patients from North India

Background

Type 1 diabetes (T1D) is a multifactorial autoimmune disorder where interaction and integration of immune response genes along with environmental factors play a role in autoimmune destruction of the insulin producing Pancreatic Beta cells.

Methodology/Principal Findings

We have studied four single nucleotide polymorphisms (FokI site in Exon 2, BsmI and ApaI sites in Intron 8 and TaqI site in exon 9) in the vitamin D receptor (VDR) gene using PCR-RFLP and HLA-DRB1 alleles using PCR and hybridization with sequence specific oligonucleotide probes and studied their interaction using LD based statistics for non-linked loci followed by sequence analysis of the vitamin D response element (VDRE) present in the promoter region of HLA-DRB1*0301. Haplotypes, constructed using SHEsis program for four single nucleotide polymorphisms in the VDR gene, were studied for their interaction with HLA-DRB1 alleles in 233 T1D patients and 191 healthy controls from North India. A significant increase of haplotypes FBAt and fBAT (p<0.02, OR = 1.44 and p<0.002, OR = 3.23 respectively) was observed in the patients. Both the haplotypes FBAt and fBAT were significantly increased in male patients with age at onset less than 18 years; however, fBAT was significantly increased in female patients irrespective of their age at onset. LD based statistics showed significant interaction between the high producer F and T alleles with HLA-DRB1*0301. F and T alleles of VDR have been shown to contribute to VDR mRNA independently. The promoter sequence analysis of HLA-DRB1*0301 showed presence of VDRE involved in higher expression of HLA-DRB1*030, which was confirmed by flow cytometry and real time PCR analysis.

Conclusions/Significance

These data suggest that the interaction between VDR and HLA alleles is mediated by VDRE present in the promoter region of HLA-DRB1*0301 allele, which may be detrimental for the manifestation of T1D in the absence of 1,25-(OH)₂D₃ in early childhood due to poor expression of DRB1*0301 in the thymus resulting in autoimmunity.     

Effect of probiotic fermented milk and chlorophyllin on gene expressions and genotoxicity during AFB₁-induced hepatocellular carcinoma

The aim of this study was to investigate the chemopreventive effect of probiotic fermented milk and chlorophyllin on aflatoxin B? (AFB?) induced hepatocellular carcinoma. In vivo trials were conducted on 200 Wistar rats allocated to eight groups. Rats in the positive control group were given intraperitoneal injection of aflatoxin B? at 450 μg/kg body weight twice a week for 6 weeks. The rats were sacrificed and dissected at 25th week of the experiment, and comet assay was carried out in hepatic cells to assess the genotoxicity or DNA damage. The tumour incidence was decreased by approximately one-third than AFB? control group. The expression of c-myc bax, bcl-2, cyclin D1, p53 and rasp-21 genes was also studied. A significant (P<0.05) reduction in DNA damage was observed in probiotic fermented milk with chlorophyllin group as compared to aflatoxin B? control group. The c-myc, bcl-2, cyclin D1 and rasp-21 level was found to be highest in AFB? control group as compared to the treatment group. The results advocate the enhanced protective potential of probiotic fermented milk and chlorophyllin against AFB?-induced molecular alterations in hepatic cells during carcinogenesis.     

Nuclear DNA assay of the wild endangered medicinal and aromatic Indian Himalayan Valeriana jatamansi germplasm with multiple DNA markers: implications for genetic enhancement,domestication and ex situ conservation

Population genetic analysis in the important endangered medicinal and aromatic plant species, Valeriana jatamansi, provided, first time, insights into the identification of novel sources of genetic variation as an aid for improvement and domestication, and for optimizing conservation strategies. The 75 genotypes of V. jatamansi were collected from 36 locations across northeast to northwest Indian Himalayas of ~1,000 km, harbouring variable climatic and ecological conditions and rugged rocky terrain. The known protocols for DNA extraction failed to yield quality DNA in good quantity. A new protocol was standardized for this purpose. All the three (RAPD, ISSR, AFLP) DNA markers were successful in detecting polymorphism in V. jatamansi genotypes, and the ISSR marker, vis-à-vis RAPD and AFLP markers, generated the highest level of polymorphism. The RAPD, ISSR and AFLP fingerprints with 23 and 15 primers and 8 primer combinations, respectively, revealed 85.8, 89.0 and 67.7 % polymorphism among 141, 91 and 37 genetic loci amplified from the 75 genotypes, respectively. The AMOVA analysis of AFLP (55.0, 8.3, 36.7 %), RAPD (57.4, 11.9, 30.6 %) and ISSR (76.0, 4.8, 19.1 %) data indicated that more variation existed in differences in genotypes within populations than between populations within a region and between regions, respectively. The present comprehensive input will assist in effective management and (or) devising conservation strategies of this important medicinal plant species. This study reports the start of a molecular biology programme targeting nuclear genome of V. jatamansi, the genetics of which is very little known.     

High Resolution T1ρ Mapping of In Vivo Human Knee Cartilage at 7T

Purpose

Spin lattice relaxation time in rotating frame (T1ρ) mapping of human knee cartilage has shown promise in detecting biochemical changes during osteoarthritis. Due to higher field strength, MRI at 7T has advantages in term of SNR compared to clinical MR scanners and this can be used to increase in image resolution. Objective of current study was to evaluate the feasibility of high resolution T1ρ mapping of in vivo human knee cartilage at 7T MR scanner.

Materials and Methods

In this study we have used a T1ρ prepared GRE pulse sequence for obtaining high resolution (in plan resolution  = 0.2 mm²) T1ρ MRI of human knee cartilage at 7T. The effect of a global and localized reference frequency and reference voltage setting on B₀, B₁ and T1ρ maps in cartilage was evaluated. Test-retest reliability results of T1ρ values from asymptomatic subjects as well as T1ρ maps from abnormal cartilage of two human subjects are presented. These results are compared with T1ρ MRI data obtained from 3T.

Results

Our approach enabled acquisition of 3D-T1ρ data within allowed SAR limits at 7T. SNR of cartilage on T1ρ weighted images was greater than 90. Off-resonance effects present in the cartilage B₀, B₁ and T1ρ maps obtained using global shim and reference frequency and voltage setting, were reduced by the proposed localized reference frequency and voltage setting. T1ρ values of cartilage obtained with the localized approach were reproducible. Abnormal knee cartilage showed elevated T1ρ values in affected regions. T1ρ values at 7T were significantly lower (p<0.05) compared to those obtained at 3T.

Conclusion

In summary, by using proposed localized frequency and voltage setting approach, high-resolution 3D-T1ρ maps of in vivo human knee cartilage can be obtained in clinically acceptable scan times (<30 min) and SAR constraints, which provides the ability to characterize cartilage molecular integrity.