Universal Plant DNA Barcode Loci May Not Work in Complex Groups: A Case Study with Indian Berberis Species

BACKGROUND: The concept of DNA barcoding for species identification has gained considerable momentum in animals because of fairly successful species identification using cytochrome oxidase I (COI). In plants, matK and rbcL have been proposed as standard barcodes. However, barcoding in complex genera is a challenging task. METHODOLOGY AND PRINCIPAL FINDINGS: We investigated the species discriminatory power of four reportedly most promising plant DNA barcoding loci (one from nuclear genome--ITS, and three from plastid genome--trnH-psbA, rbcL and matK) in species of Indian Berberis L. (Berberidaceae) and two other genera, Ficus L. (Moraceae) and Gossypium L. (Malvaceae). Berberis species were delineated using morphological characters. These characters resulted in a well resolved species tree. Applying both nucleotide distance and nucleotide character-based approaches, we found that none of the loci, either singly or in combinations, could discriminate the species of Berberis. ITS resolved all the tested species of Ficus and Gossypium and trnH-psbA resolved 82% of the tested species in Ficus. The highly regarded matK and rbcL could not resolve all the species. Finally, we employed amplified fragment length polymorphism test in species of Berberis to determine their relationships. Using ten primer pair combinations in AFLP, the data demonstrated incomplete species resolution. Further, AFLP analysis showed that there was a tendency of the Berberis accessions to cluster according to their geographic origin rather than species affiliation. CONCLUSIONS/SIGNIFICANCE: We reconfirm the earlier reports that the concept of universal barcode in plants may not work in a number of genera. Our results also suggest that the matK and rbcL, recommended as universal barcode loci for plants, may not work in all the genera of land plants. Morphological, geographical and molecular data analyses of Indian species of Berberis suggest probable reticulate evolution and thus barcode markers may not work in this case

In vitro proliferation of shoots and regeneration of cotton

Shoot proliferation from different explants of several Indian cultivars of cotton was studied in culture. Cotyledonary nodes taken along with the shoot apex of seedlings produced multiple shoots on modified MS nutrient agar supplemented with cytokinins. 6-Benzyladenine was most effective in inducing growth of multiple shoots. Explants of several genotypes formed organogenic masses that differentiated to secondary shoots on repeated subculture. The isolated shoots were rooted on basal medium supplemented with naphthaleneacetic acid and were transferred to soil after acclimatization

Expression of δ-endotoxin Cry1EC from an inducible promoter confers insect protection in peanut (Arachis hypogaea L.) plants

BACKGROUND:Spodoptera litura (F.) is a polyphagous foliage insect and a major pest on peanut (Arachis hypogaea L.). Constitutive expression of d-endotoxin Cry1EC gives protection against S. litura, as reported earlier. In this study, insect bites and salicylic acid induced high-level expression of Cry1EC was achieved in peanut. In order to achieve this, the expression of pathogenesis responsive promoter PR-1a was enhanced by placing it downstream of the CaMV35S promoter in the pCAMBIA 1300 backbone. The resultant promoter CaMV35S(r)PR-1a expressed a high level of insecticidal d-endotoxin Cry1EC. The Gus expression under the control of CaMV35S(r)PR-1a served as a convenient marker for evaluation of promoter response to different treatments. RESULTS: Transgenic events that showed a very low level of uninduced expression and no expression in seeds were selected. The Cry1EC expression in leaves increased nearly eightfold in the selected event, following induction by salicylic acid. Both the salicylic-acid-treated and the S. litura-bitten leaves showed the highest expression after 2 days. Leaves from salicylic-acid-induced transgenic plants caused 100% mortality of S. litura at all stages of larval development. CONCLUSION: The results suggest that high expression of inducible promoters provides a good strategy for the development of safer transgenic food and feed crops

Plants as bioreactors for the production of vaccine antigens

Plants have been identified as promising expression systems for commercial production of vaccine antigens. In phase I clinical trials several plant-derived vaccine antigens have been found to be safe and induce sufficiently high immune response. Thus, transgenic plants, including edible plant parts are suggested as excellent alternatives for the production of vaccines and economic scale-up through cultivation. Improved understanding of plant molecular biology and consequent refinement in the genetic engineering techniques have led to designing approaches for high level expression of vaccine antigens in plants. During the last decade, several efficient plant-based expression systems have been examined and more than 100 recombinant proteins including plant-derived vaccine antigens have been expressed in different plant tissues. Estimates suggest that it may become possible to obtain antigen sufficient for vaccinating millions of individuals from one acre crop by expressing the antigen in seeds of an edible legume, like peanut or soybean. In the near future, a plethora of protein products, developed through 'naturalized bioreactors' may reach market. Efforts for further improvements in these technologies need to be directed mainly towards validation and applicability of plant-based standardized mucosal and edible vaccines, regulatory pharmacology, formulations and the development of commercially viable GLP protocols. This article reviews the current status of developments in the area of use of plants for the development of vaccine antigens

Effect of light intensity on <i>in vitro </i>multiple shoot induction and regeneration of cotton (<i>Gossypium hirsutum </i>L. cv Khandawa-2)

399-401Cotyledonary nodes taken alongwith shoot apex from seedlings of cotton (G. hirsutum) proliferated into shoots on nutrient agar medium supplemented with cytokinins. In the presence of optimal plant growth regulators, low light intensity enhanced the number of shoots initiated per explant in cotton. An average of 33.5±2.9 shoots were obtained from a single explant cultured for 8 weeks which is about four fold higher than the values reported in earlier protocols. The isolated shoots were rooted on nutrient agar medium supplemented with α-naphthalene acetic acid and transferred to soil after acclimatization. Regenerated plants were morphologically identical to the seed-germinated plants and were fertile

Rabies glycoprotein fused with B subunit of cholera toxin expressed in tobacco plants folds into biologically active pentameric protein

The pentameric B subunit of cholera toxin (CtxB) is an efficient mucosal adjuvant for vaccines. We report the expression of a chimeric protein comprising the synthetic cholera toxin B subunit fused at its C-terminal with rabies surface glycoprotein (G protein) in tobacco plants. The ⋍80.3 kDa fusion polypeptide expressed at 0.4% of the total soluble protein in leaves of the selected transgenic lines. The fusion protein formed a ⋍403 kDa pentameric protein which was functionally active in binding to GM1 receptor. The plant-made protein had a higher affinity for GM1 receptor than the native bacterial CtxB. The pentameric fusion protein was recognized by the anti-cholera toxin as well as anti-rabies antibodies. Its immuno-protective ability against rabies remains to be examined

BECLIN1 from Arabidopsis thaliana under the generic control of regulated expression systems, a strategy for developing male sterile plants

Induction of male sterility followed by successful outcrossing is a prerequisite for hybrid seed production. In this article, we have identified a novel use of the BECLIN 1 gene of Arabidopsis, in inducing male sterility in plants, when expressed in the anther tapetum of tobacco. We also report a stringently regulated and high-level expression of the desired gene in tapetum by using a two-component transcription regulation system. The tapetum-specific, two-component transcription system utilizes the TGTA-TBPm<SUB>3</SUB> complementation principle that has been demonstrated by us earlier. We also report a glucocorticoid-dependent expression of AtBECLIN 1 in tapetum, thereby developing glucocorticoid-inducible male sterility in plants

Enhanced whitefly resistance in transgenic tobacco plants expressing double stranded RNA of v-ATPase A gene.

BACKGROUND: Expression of double strand RNA (dsRNA) designed against important insect genes in transgenic plants have been shown to give protection against pests through RNA interference (RNAi), thus opening the way for a new generation of insect-resistant crops. We have earlier compared the efficacy of dsRNAs/siRNAs, against a number of target genes, for interference in growth of whitefly (Bemisia tabaci) upon oral feeding. The v-ATPase subunit A (v-ATPaseA) coding gene was identified as a crucial target. We now report the effectiveness of transgenic tobacco plants expressing siRNA to silence v-ATPaseA gene expression for the control of whitefly infestation. METHODOLOGY/PRINCIPAL FINDINGS: Transgenic tobacco lines were developed for the expression of long dsRNA precursor to make siRNA and knock down the v-ATPaseA mRNA in whitefly. Molecular analysis and insecticidal properties of the transgenic plants established the formation of siRNA targeting the whitefly v-ATPaseA, in the leaves. The transcript level of v-ATPaseA in whiteflies was reduced up to 62% after feeding on the transgenic plants. Heavy infestation of whiteflies on the control plants caused significant loss of sugar content which led to the drooping of leaves. The transgenic plants did not show drooping effect. CONCLUSIONS/SIGNIFICANCE: Host plant derived pest resistance was achieved against whiteflies by genetic transformation of tobacco which generated siRNA against the whitefly v-ATPaseA gene. Transgenic tobacco lines expressing dsRNA of v-ATPaseA, delivered sufficient siRNA to whiteflies feeding on them, mounting a significant silencing response, leading to their mortality. The transcript level of the target gene was reduced in whiteflies feeding on transgenic plants. The strategy can be taken up for genetic engineering of plants to control whiteflies in field crops

The extent of genetic diversity among Vanilla species: comparative results for RAPD and ISSR

Vanilla is a large genus of about 110 species in the orchid family (Orchidaceae), including the species Vanilla planifolia from which commercial vanilla flavoring is derived. Since most species of vanilla are considered rare and endangered there is an urgent need to conserve them through genetic analysis and propagation/conservation studies on this crop. The present study investigated the genetic diversity among nine leafy-and leaf-less Vanilla species employing 30 decamer RAPD primers and 10 ISSR primers. The species under study were diverse and displayed a range of variability (0-66% and 0-81% for RAPD and ISSR, respectively). A total of 154 RAPD polymorphic markers (83.24%, h=0.378) and 93 ISSR polymorphic markers (86.11%, h=0.363) were used to generate a genetic similarity matrix followed by the cluster analysis. Specific groupings were revealed by each cluster analysis with slight variation between two different markers. Among the nine species studied, V. planifolia, Vanilla aphylla and Vanilla tahitensis revealed very low level of variation within their collections, thus indicating a narrow genetic base. The large genetic distance of Vanilla andamanica from other species suggests its different origin. A close genetic affinity was observed between the pairs V. planifolia, V. tahitensis and Vanilla albida, V. aphylla. These are the first comparative results for RAPD and ISSR reporting inter-relationship among nine cultivated, wild and hybrid Vanilla species

Comparative transcriptome analysis of Gossypium hirsutum L. in response to sap sucking insects: aphid and whitefly

BACKGROUND: Cotton (Gossypium hirsutum L.) is a major fiber crop that is grown worldwide; it faces extensive damage from sap-sucking insects, including aphids and whiteflies. Genome-wide transcriptome analysis was performed to understand the molecular details of interaction between Gossypium hirsutum L. and sap-sucking pests, namely Aphis gossypii (Aphid) and Bemisia tabacci (Whiteflies). Roche’s GS-Titanium was used to sequence transcriptomes of cotton infested with aphids and whiteflies for 2 h and 24 h. RESULTS: A total of 100935 contigs were produced with an average length of 529 bp after an assembly in all five selected conditions. The Blastn of the non-redundant (nr) cotton EST database resulted in the identification of 580 novel contigs in the cotton plant. It should be noted that in spite of minimal physical damage caused by the sap-sucking insects, they can change the gene expression of plants in 2 h of infestation; further change in gene expression due to whiteflies is quicker than due to aphids. The impact of the whitefly 24 h after infestation was more or less similar to that of the aphid 2 h after infestation. Aphids and whiteflies affect many genes that are regulated by various phytohormones and in response to microbial infection, indicating the involvement of complex crosstalk between these pathways. The KOBAS analysis of differentially regulated transcripts in response to aphids and whiteflies indicated that both the insects induce the metabolism of amino acids biosynthesis specially in case of whiteflies infestation at later phase. Further we also observed that expression of transcript related to photosynthesis specially carbon fixation were significantly influenced by infestation of Aphids and Whiteflies. CONCLUSIONS: A comparison of different transcriptomes leads to the identification of differentially and temporally regulated transcripts in response to infestation by aphids and whiteflies. Most of these differentially expressed contigs were related to genes involved in biotic, abiotic stresses and enzymatic activities related to hydrolases, transferases, and kinases. The expression of some marker genes such as the overexpressors of cationic peroxidase 3, lipoxygenase I, TGA2, and non-specific lipase, which are involved in phytohormonal-mediated plant resistance development, was suppressed after infestation by aphids and whiteflies, indicating that insects suppressed plant resistance in order to facilitate their infestation. We also concluded that cotton shares several pathways such as phagosomes, RNA transport, and amino acid metabolism with Arabidopsis in response to the infestation by aphids and whiteflies