Stress-inducible expression of barley Hva1 gene in transgenic mulberry displays enhanced tolerance against drought, salinity and cold stress

Coping with different kinds of biotic and abiotic stresses is the foundation of sustainable agriculture. Although conventional breeding and marker-assisted selection are being employed in mulberry (Morus indica L.) to develop better varieties, nonetheless the longer time periods required for these approaches necessitates the use of precise biotechnological approaches for sustainable agriculture. In an attempt to improve stress tolerance of mulberry, an important plant of the sericulture industry, an encoding late embryogenesis abundant gene from barley (HVA1) was introduced into mulberry plants by Agrobacterium-mediated transformation. Transgenic mulberry with barley Hva1 under a constitutive promoter actin1 was shown to enhance drought and salinity tolerance. Here, we report that overexpression of barley Hva1 also confers cold tolerance in transgenic mulberry. Further, barley Hva1 gene under control of a stress-inducible promoter rd29A can effectively negate growth retardation under non-stress conditions and confer stress tolerance in transgenic mulberry. Transgenic lines display normal morphology to enhanced growth and an increased tolerance against drought, salt and cold conditions as measured by free proline, membrane stability index and PSII activity. Protein accumulation was detected under stress conditions confirming inductive expression of HVA1 in transgenics. Investigations to assess stress tolerance of these plants under field conditions revealed an overall better performance than the non-transgenic plants. Enhanced expression of stress responsive genes such as Mi dnaJ and Mi 2-cysperoxidin suggests that Hva1 can regulate downstream genes associated with providing abiotic stress tolerance. The investigation of transgenic lines presented here demonstrates the acquisition of tolerance against drought, salt and cold stress in plants overexpressing barley Hva1, indicating that Arabidopsis rd29A promoter can function in mulberry.     

Safety evaluation of genetically modified mustard (V4) seeds in terms of allergenicity: Comparison with native crop

Genetically modified (GM) mustard line (V4) with increased carotenoid content was compared with native mustard to find the difference in allergenic potential, if any. Simulated gastric fluid (SGF) digestibility of crude protein extract from GM as well as its native counterpart mustard crop was envisaged to understand the intended or unintended changes in GM crop along with IgE immunoblotting. BALB/c mice were used as model for allergenicity studies for monitoring total and specific IgE, specific IgG1, histamine level, histopathology, and systemic anaphylaxis score. Allergenicity of mustard was checked in humans by clinical history, skin prick test and IgE levels. Similar results were evident by significant increase in total IgE, specific IgE, IgG1, histamine levels, in GM and native mustard in comparison to control group. Prominent anaphylactic symptoms (score 2: 60%; score 3: 20%; score 4: 20% in native mustard and score 2: 40%; score 3: 40%; score 4: 20% in GM mustard) and eruptive histopathological changes were observed in both GM and native mustard when compared with controls. One protein of approximately 16 kDa was found stable up to 1 h in both GM as well as non GM mustard. IgE immunoblotting detected three protein components of approximately 29, 24 and 16 kDa in both GM and non GM varieties. Collectively, our data demonstrate substantially equivalent allergic responses against GM as well as its native counterpart. Therefore, the GM mustard may be as safe as its native counterpart with reference to allergenic responses.     

ISSR assay for ascertaining genetic fidelity of micropropagated plants of apple rootstock Merton 793

With the current trends in high density plantations of fruit trees, numerous clonal rootstocks of apple have been developed through various breeding programs. Among them, Merton 793 is the most popular in India because of the desirable traits of vigorous growth and resistance to woolly apple aphid and collar rot. The planting material of this rootstock cannot be multiplied at a desirable rate by means of conventional vegetative propagation methods, so micropropagation techniques are being explored to augment scarce planting material. Large number of plants can be produced in vitro under aseptic conditions, but there is always a danger of producing somaclonal variants by tissue culture technology. Thus, it is advisable to check the clonal fidelity of in vitro raised plants, especially of perennials prior to their field transplantation. The genetic stability of in vitro raised plants of apple rootstock Merton 793, multiplied through enhanced axillary bud proliferation up to 22 subculture passages, was tested by intersimple sequence repeat (ISSR) assay. Of 24 ISSR primers screened, 15 primers produced clear reproducible bands, resulting in a total of 134 distinct bands with an average of 8.9 bands per primer. Apple rootstock MM 111 and scion Jonathan, taken as outliers with tissue culture-raised progenies of Merton 793, ruled out the possibility that the invariant banding pattern occurred because of inefficiency of ISSR primers in detecting variations. The homogenous amplification profile observed for all the micropropagated plants compared to the donor plant confirmed the clonal fidelity of the tissue culture-raised Merton 793 plants. This suggests that axillary bud multiplication is the safest mode for multiplication of true-to-type plants. This is the first study that evaluates the applicability of ISSR markers in establishing clonal fidelity of tissue culture-raised apple plants.     

Impaired Male Meiosis due to Irregular Synapsis Coupled with Cytomixis in a New Diploid Cytotype of Dianthus angulatus (Caryophyllaceae) from Indian Cold Deserts

Dianthus angulatus (Caryophyllaceae) is cytologically examined here for the first time for the area of India. The diploid chromosome count of 2n?=?30, ascertained here, represents a new cytotype, supplementing the earlier report of a hexaploid cytotype with 2n?=?90 from outside of India. We report here the occurrence of two plants showing impaired meiosis due to irregular synapsis and cytomixis collected from Kinnaur district, Himachal Pradesh (India). The other plants of this species collected in Lahaul-Spiti region showed normal male meiosis (n?=?15) with a high (95%?100%) pollen fertility and normal seed set, and they reproduce sexually. Irregular synapsis in two plants from the Kinnaur region is characterized by the complete absence of chromosome pairing and the presence of 30 univalents at diakinesis and meta-anaphase. In addition, other meiotic irregularities were found, such as unoriented chromosomes, laggards, precocious movements of univalents at anaphase-I and micronuclei at telophase. Microsporogenesis was also abnormal, resulting in the formation of monads, dyads, triads, polyads, and tetrads with micronuclei. The occurrence of intra- and intermicrosporal chromatin material transfer during microsporogenesis was observed, which is a rather rarely observed phenomenon. The synaptic irregularities coupled with chromatin transfer in these plants seem to be responsible for the high pollen sterility (38%?42%) and heterogeneously sized pollen grains. In these plants no seeds were set, and plants reproduced vegetatively through root suckers.     

Marker-free site-specific gene integration in rice based on the use of two recombination systems

Transgene integration mediated by heterologous site-specific recombination (SSR) systems into the dedicated genomic sites has been demonstrated in a few different plant species. This approach of plant transformation generates a precise site-specific integration (SSI) structure consisting of a single copy of the transgene construct. As a result, stable transgene expression correlated with promoter strength and gene copy number is observed among independent transgenic lines and faithfully transmitted through subsequent generations. Site-specific integration approaches use selectable marker genes, removal of which is necessary for the implementation of this approach as a biotechnology application. As SSR systems are also excellent tools for excising marker genes from transgene locus, a molecular strategy involving gene integration followed by marker excision, each mediated by a distinct recombination system, was earlier proposed. Experimental validation of this approach is the focus of this work. Using FLPe-FRT system for site-specific gene integration and heat-inducible Cre-lox for marker gene excision, marker-free SSI lines were developed in the first generation itself. More importantly, progeny derived from these lines inherited the marker-free locus, indicating efficient germinal transmission. Finally, as the transgene expression from SSI locus was not altered upon marker excision, this method is suitable for streamlining the production of marker-free SSI lines.     

Prokaryotic gene finding based on physicochemical characteristics of codons calculated from molecular dynamics simulations

An ab initio model for gene prediction in prokaryotic genomes is proposed based on physicochemical characteristics of codons calculated from molecular dynamics (MD) simulations. The model requires a specification of three calculated quantities for each codon: the double-helical trinucleotide base pairing energy, the base pair stacking energy, and an index of the propensity of a codon for protein-nucleic acid interactions. The base pairing and stacking energies for each codon are obtained from recently reported MD simulations on all unique tetranucleotide steps, and the third parameter is assigned based on the conjugate rule previously proposed to account for the wobble hypothesis with respect to degeneracies in the genetic code. The third interaction propensity parameter values correlate well with ab initio MD calculated solvation energies and flexibility of codon sequences as well as codon usage in genes and amino acid composition frequencies in ∼175,000 protein sequences in the Swissprot database. Assignment of these three parameters for each codon enables the calculation of the magnitude and orientation of a cumulative three-dimensional vector for a DNA sequence of any length in each of the six genomic reading frames. Analysis of 372 genomes comprising ∼350,000 genes shows that the orientations of the gene and nongene vectors are well differentiated and make a clear distinction feasible between genic and nongenic sequences at a level equivalent to or better than currently available knowledge-based models trained on the basis of empirical data, presenting a strong support for the possibility of a unique and useful physicochemical characterization of DNA sequences from codons to genomes.     

Use of metabolic stimulators and inhibitors for enhanced production of beta-carotene and lycopene by Blakeslea trispora NRRL 2895 and 2896

This work investigates the potential of metabolic stimulators, firstly to enhance the production of beta-carotene, and later use of inhibitors of lycopene cyclase so as to accumulate lycopene in the fermentation medium. Various non-ionic surfactants, natural oils, stimulators such as amino-acids, tricarboxylic acid cycle (TCA) intermediates, vitamin A and antibiotics were investigated for improved production of beta-carotene using the zygomycete fungus Blakeslea trispora. Span 20 at 0.2% increased the beta-carotene production from 139 mg/l to 318 mg/l. Examination of the mycelial morphology of the B. trispora with span 20 showed a shorter mycelial length, which allowed a well-dispersed growth of B. trispora. Supplementation of the medium with 1000 ppm vitamin A acetate gave highest concentration of beta-carotene (830+/-6 mg/l). Several chemical inhibitors such as imidazole, pyridine, triethylamine, piperidine, and nicotinic acid were then evaluated to block the biosynthesis at lycopene. Piperidine at 500 ppm gave a 7.76-fold improvement, and produced high titers of lycopene (775+/-5 mg/l) in a medium supplemented with vitamin A acetate.     

Molecular Marker Based Coefficient of Parentage Analysis for Establishing Distinctness in Indian Rice (Oryza sativa L) Varieties

Molecular characterization of 32 Indian rice varieties of different agro-climatic zones resulted in mean heterozygosity value of 0.622, 0.819 and 0.890 over polymorphic loci and marker index value of 1.00, 6.75 and 4.16, respectively for RAPD, ISSR and STMS primers. The three marker systems resulted in 201 polymorphic bands (94.36%) out of a total of 213 bands. The probability of a chance identical match between two varieties was very low (2.08x10¹⁰) in combined molecular marker analysis as compared to individual marker system (RAPD, 7.5 x 10^-4; ISSR, 1.5 x 10^-3 and STMS, 3.9 x 10^-6). The combined average genetic similarity for molecular markers and coefficient of parentage (COP) across all 496 pairwise combinations revealed a non-significant relationship (r = 0.215).