Praveen C. Verma

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.

Influence of inoculation of arsenic-resistant Staphylococcus arlettae on growth and arsenic uptake in Brassica juncea (L.) Czern. Var. R-46

An arsenic hypertolerant bacterium was isolated from arsenic contaminated site of West Bengal, India. The bacteria was identified as Staphylococcus arlettae strain NBRIEAG-6, based on 16S rDNA analysis. S. arlettae was able to remove arsenic from liquid media and possesses arsC gene, gene responsible for arsenate reductase activity. The biochemical profiling of the isolated strain showed that it had the capacity of producing indole acetic acid (IAA), siderophores and 1-aminocyclopropane-1-carboxylic acid (ACC) deaminase. Furthermore, an experiment was conducted to test the effect of S. arlettae inoculation on concurrent plant growth promotion and arsenic uptake in Indian mustard plant [Brassica juncea (L.) Czern. Var. R-46] when grown in arsenic spiked (5, 10 and 15 mg kg(-1)) soil. The microbial inoculation significantly (p<0.05) increased biomass, protein, chlorophyll and carotenoids contents in test plant. Moreover, as compared to the non-inoculated control, the As concentration in shoot and root of inoculated plants were increased from 3.73 to 34.16% and 87.35 to 99.93%, respectively. The experimental results show that the plant growth promoting bacteria NBRIEAG-6 has the ability to help B. juncea to accumulate As maximally in plant root, and therefore it can be accounted as a new bacteria for As phytostabilization.

Functional analysis of sucrose phosphate synthase (SPS) and sucrose synthase (SS) in sugarcane (Saccharum) cultivars

Sucrose phosphate synthase (SPS; EC 2.4.1.14) and sucrose synthase (SS; EC 2.4.1.13) are key enzymes in the synthesis and breakdown of sucrose in sugarcane. The activities of internodal SPS and SS, as well as transcript expression were determined using semi-quantitative RT-PCR at different developmental stages of high and low sucrose accumulating sugarcane cultivars. SPS activity and transcript expression was higher in mature internodes compared with immature internodes in all the studied cultivars. However, high sugar cultivars showed increased transcript expression and enzyme activity of SPS compared to low sugar cultivars at all developmental stages. SS activity was higher in immature internodes than in mature internodes in all cultivars; SS transcript expression showed a similar pattern. Our studies demonstrate that SPS activity was positively correlated with sucrose and negatively correlated with hexose sugars. However, SS activity was negatively correlated with sucrose and positively correlated with hexose sugars. The present study opens the possibility for improvement of sugarcane cultivars by increasing expression of the respective enzymes using transgene technology.

Agrobacterium rhizogenes -mediated transformation of Picrorhiza kurroa Royle ex Benth.: establishment and selection of superior hairy root clone

A protocol for induction and establishment of Agrobacterium rhizogenes-mediated hairy root cultures of Picrorhiza kurroa was developed through optimization of the explant type and the most suitable bacterial strain. The infection of leaf explants with the LBA9402 strain resulted in the emergence of hairy roots at 66.7% relative transformation frequency. Nine independent, opine and TL-positive hairy root clones were studied for their growth and specific glycoside (i.e., kutkoside and picroside I) productivities at different growth phases. Biosynthetic potentials for the commercially desirable active constituents have been expressed by all the tested hairy root clones, although distinct inter-clonal variations could be noted in terms of their quantity. The yield potentials of the 14-P clone, both in terms of biomass as well as individual glycoside contents (i.e., kutkoside and picroside I), superseded that of all other hairy root clones along with the non-transformed, in vitro-grown control roots of P. kurroa. The present communication reports the first successful establishment, maintenance, growth and selection of superior hairy root clone of Picrorhiza kurroa with desired phyto-molecule production potential, which can serve as an effective substitute to its roots and thereby prevent the indiscriminate up-rooting and exploitation of this commercially important, endangered medicinal plant species.

Isolation and characterization of Staphylococcus sp. strain NBRIEAG-8 from arsenic contaminated site of West Bengal

Arsenic contaminated rhizospheric soils of West Bengal, India were sampled for arsenic resistant bacteria that could transform different arsenic forms. Staphylococcus sp. NBRIEAG-8 was identified by16S rDNA ribotyping, which was capable of growing at 30,000xa0mgxa0l−1 arsenate [As(V)] and 1,500xa0mgxa0l−1 arsenite [As(III)]. This bacterial strain was also characterized for arsenical resistance (ars) genes which may be associated with the high-level resistance in the ecosystems of As-contaminated areas. A comparative proteome analysis was conducted with this strain treated with 1,000xa0mgxa0l−1 As(V) to identify changes in their protein expression profiles. A 2D gel analysis showed a significant difference in the proteome of arsenic treated and untreated bacterial culture. The change in pH of cultivating growth medium, bacterial growth pattern (kinetics), and uptake of arsenic were also evaluated. After 72xa0h of incubation, the strain was capable of removing arsenic from the culture medium amended with arsenate and arsenite [12% from As(V) and 9% from As(III)]. The rate of biovolatilization of As(V) was 23% while As(III) was 26%, which was determined indirectly by estimating the sum of arsenic content in bacterial biomass and medium. This study demonstrates that the isolated strain, Staphylococcus sp., is capable for uptake and volatilization of arsenic by expressing ars genes and 8 new upregulated proteins which may have played an important role in reducing arsenic toxicity in bacterial cells and can be used in arsenic bioremediation.

Rose-scented geranium (Pelargonium sp.) generated by Agrobacterium rhizogenes mediated Ri-insertion for improved essential oil quality

Transgenic plants of rose-scented geranium (Pelargonium graveolens cv. Hemanti) have been produced from Agrobacterium rhizogenes (strains A4 and LBA9402) mediated hairy root cultures. Amongst the explants tested, leaves were most responsive followed by the petioles and internodal segments, respectively. The A4 strain performed better for all the three explants both in terms of frequency of response and time requirement for hairy root induction. Transgenic shoots could be obtained by spontaneous regeneration without intervening callus phase amongst 16% and 12% root lines of A4 and LBA 9402 origin, respectively, or they were induced in 29% and 22% hairy root lines of A4 and LBA9402 origin, respectively, with different hormonal supplementation. These transgenic plants showed 30% survival as against 90% of their control under the confined environment of glasshouse. The transgenic plants were of similar morphotype having increased branching, higher number of leaves with increased dentations, short and round stature, highly branched root system and absence of leaf wrinkling. These transgenic plants showed opine positive results even after 5xa0months of their transfer to the glasshouse. The essential oil compositions of 81% of these transgenics were qualitatively similar to that of the wild type parent. However, two transgenic plants (LZ-3 and 14TG) showed increase in concentrations of geraniol and geranyl esters signifying improved oil quality with respect to the citronellol:geraniol ratio. These two oils having better olfactory value represent an improvement over that of the wild type parent from the commercial point of view.

Brevundimonas diminuta mediated alleviation of arsenic toxicity and plant growth promotion in Oryza sativa L.

Arsenic (As), a toxic metalloid adversely affects plant growth in polluted areas. In the present study, we investigated the possibility of improving phytostablization of arsenic through application of new isolated strain Brevundimonas diminuta (NBRI012) in rice plant [Oryza sativa (L.) Var. Sarju 52] at two different concentrations [10ppm (low toxic) and 50ppm (high toxic)] of As. The plant growth promoting traits of bacterial strains revealed the inherent ability of siderophores, phosphate solubilisation, indole acetic acid (IAA), 1-aminocyclopropane-1-carboxylic acid (ACC) deaminase production which may be associated with increased biomass, chlorophyll and MDA content of rice and thereby promoting plant growth. The study also revealed the As accumulation property of NBRI012 strain which could play an important role in As removal from contaminated soil. Furthermore, NBRI012 inoculation significantly restored the hampered root epidermal and cortical cell growth of rice plant and root hair elimination. Altogether our study highlights the multifarious role of B. diminuta in mediating stress tolerance and modulating translocation of As in edible part of rice plant.

Transcript expression and soluble acid invertase activity during sucrose accumulation in sugarcane

Soluble acid invertase (SAI, EC 3.2.1.26), catalyzes the hydrolysis of sucrose into hexose sugars, and it has been considered a key enzyme for carbohydrate metabolism. In the present study, the activity of SAI enzyme was determined to establish a correlation between the change in transcript levels and enzyme activity in high and low sugar accumulating sugarcane cultivars, in various internodal tissues at different developmental stages. A decrease in SAI activity and transcript levels was observed with age, during all the developmental stages in both the cultivars. A negative correlation between SAI activity and sucrose content was observed in mature and immature internodes; however, there was a positive correlation between SAI activity and content of hexose sugars. These results imply that SAI plays a crucial role in sucrose partitioning in various intermodal tissues in high and low sugar cultivars. In addition to this, the changes in enzyme activity also resulted in changes in transcript level.

Soil fungi for mycoremediation of arsenic pollution in agriculture soils.

Soil arsenic (As) contamination of food‐chains and public health can be mitigated through fungal bioremediation. To enumerate culturable soil fungi, soils were collected from the As‐contaminated paddy fields (3–35 mg kg−1) of the middle Indo‐Gangetic Plains.

Expression of Withania somnifera Steroidal Glucosyltransferase gene Enhances Withanolide Content in Hairy Roots

In Withania somnifera, sterol molecules of immense medicinal value are diversified by means of glycosylation. Identifying sterol glycosyltransferases provides an imperative insight of diverse sterol modifications, thereby helping to comprehend the underlying plant mechanisms. In the present study, one of the W. somnifera sterol glycosyltransferase-4 (Ws-Sgtl4) gene was transformed into the W. somnifera leaf explant through Agrobacterium rhizogene. Transformed W. Somnifera Ws-Sgtl4 leaf explants were subjected to hairy root induction and analyzed for biomass accumulation. The analysis of Ws-Sgtl4 gene expression was performed at different time exposures with the application of salicylic acid and methyl jasmonate. The elicitation of W. somnifera hairy root expressing the Ws-Sgtl4 gene was also evaluated for the enhancement if any, in the total withanolide yield as well as the withanolides-A contents. The results suggested that Ws-Sgtl4 gene expression enhanced the production of total withanolide yield and withanolides-A in the hairy root culture of W. somnifera in the response to the elicitors.