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The clustered, regularly interspaced, short palindromic repeats (CRISPR) and CRISPR-associated protein (Cas) system has been used as an efficient tool for genome editing. We report the application of CRISPR-Cas–mediated genome editing to wheat (Triticum aestivum), the most important food crop plant with a very large and complex genome. The mutations were targeted in the inositol oxygenase (inox) and phytoene desaturase (pds) genes using cell suspension culture of wheat and in the pds gene in leaves of Nicotiana benthamiana. The expression of chimeric guide RNAs (cgRNA) targeting single and multiple sites resulted in indel mutations in all the tested samples. The expression of Cas9 or sgRNA alone did not cause any mutation. The expression of duplex cgRNA with Cas9 targeting two sites in the same gene resulted in deletion of DNA fragment between the targeted sequences. Multiplexing the cgRNA could target two genes at one time. Target specificity analysis of cgRNA showed that mismatches at the 3′ end of the target site abolished the cleavage activity completely. The mismatches at the 5′ end reduced cleavage, suggesting that the off target effects can be abolished in vivo by selecting target sites with unique sequences at 3′ end. This approach provides a powerful method for genome engineering in plants.

RNA interference (RNAi)-mediated gene silencing was explored for the control of sap-sucking pest Bemisia tabaci, commonly known as whitefly. dsRNAs and siRNAs were synthesized from five different genes - actin ortholog, ADP/ATP translocase, alpha-tubulin, ribosomal protein L9 (RPL9) and V-ATPase A subunit. A simplified insect bioassay method was developed for the delivery of ds/siRNA through the oral route, and efficacy was evaluated. ds/siRNA caused 29-97% mortality after 6 days of feeding. Each insect ingested nearly 150 nl of insect diet per day, which contained a maximum of 6 ng of RNA. Knocking down the expression of RPL9 and V-ATPase A caused higher mortality with LC50 11.21 and 3.08 microg/ml, respectively, as compared to other genes. Semi-quantitative PCR of the treated insects showed significant decrease in the level of RPL9 and V-ATPase A transcripts. siRNAs were found stable in the insect diet for at least 7 days at the room temperature. Phloem-specific expression of dsRNAs of RPL9 and V-ATPase A in transgenic plants for the protection against whiteflies might be an interesting application of this technology.

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Flavonoids synthesized by the phenylpropanoid pathway participate in myriad physiological and biochemical processes in plants. Due to the diversity of secondary transformations and the complexity of the regulation of branched pathways, single gene strategies have not been very successful in enhancing the accumulation of targeted molecules. We have expressed an Arabidopsis (Arabidopsis thaliana) transcription factor, AtMYB12, in tobacco (Nicotiana tabacum), which resulted in enhanced expression of genes involved in the phenylpropanoid pathway, leading to severalfold higher accumulation of flavonols. Global gene expression and limited metabolite profiling of leaves in the transgenic lines of tobacco revealed that AtMYB12 regulated a number of pathways, leading to flux availability for the phenylpropanoid pathway in general and flavonol biosynthesis in particular. The tobacco transgenic lines developed resistance against the insect pests Spodoptera litura and Helicoverpa armigera due to enhanced accumulation of rutin. Suppression of flavonol biosynthesis by artificial microRNA reversed insect resistance of the AtMYB12-expressing tobacco plants. Our study suggests that AtMYB12 can be strategically used for developing safer insect pest-resistant transgenic plants.

BackgroundThe 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 FindingsWe 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/SignificanceWe 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.

BackgroundExpression 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 FindingsTransgenic 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/SignificanceHost 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.