<span style="font-size:15.0pt;font-family: "Times New Roman";mso-fareast-font-family:"Times New Roman";mso-bidi-font-family: "Times New Roman";mso-ansi-language:EN-GB;mso-fareast-language:EN-US; mso-bidi-language:HI" lang="EN-GB">Potential of RAPD and ISSR markers for assessing genetic diversity among <i>Stevia rebaudiana</i> Bertoni accessions</span>

95-100<span style="font-size:9.0pt;font-family: " times="" new="" roman";mso-fareast-font-family:"times="" roman";mso-bidi-font-family:="" "times="" roman";mso-ansi-language:en-gb;mso-fareast-language:en-us;="" mso-bidi-language:hi"="" lang="EN-GB">In the present study, genetic diversity among 16 collections of Stevia rebaudiana Bertoni was assessed to compare the efficiency of two-marker systems over one marker using RAPD and ISSR markers. How well these two marker systems discriminated the Stevia collections was compared. In fact, all the Stevia accessions included in the present study have been growing under different altitudes, ranging 250 to 2250 m at different geographical conditions. The 22 selected RAPD primers yielded 66 scorable bands, 54 of which were polymorphic (81.8%). While the 23 selected ISSR primers amplified 49 bands, 44 of which were polymorphic (89.8%). The genetic similarity among the accessions varied from 0.365 to 0.887 when pooled data from ISSR and RAPD were analyzed through UPGMA. The analysis of combined data set of both the techniques clustered the genotypes, based on their geographic locations. Both genotypes A & B collected from Solan (Himachal Pradesh) were known to be contrasting for different levels of rebaudioside-A and stevioside and have been singled out by all the dendrograms generated by the techniques separately. High polymorphism obtained indicates that both techniques are efficient for evaluating genetic diversity in S. rebaudiana, though ISSR yielded more polymorphism. Usually, a combination of diverse marker types is recommended to provide an accurate assessment by comparison of results.</span

Exploitation of <i>Malus</i> ESTs for development of SSR markers after <i>in silico</i> analysis

The domestic apple (Malus X domestica) has been extensively characterized for EST sequences and currently 330181 ESTs are available in NCBI. Although this is a valuable resource for marker development but the redundancy in sequences makes the mining out of unique candidates for marker designing cumbersome. Keeping this in view, the present study was undertaken with the aim to remove the data redundancy in apple ESTs and then to develop SSR markers. The EST sequences were assembled into a non – redundant set of 5619 contigs and 59343 singletons (total sequences 64962), indicating 80% reduction in data redundancy. These 64962 sequences were then used to mine out SSR motifs. 4575 EST-SSRs were detected, with dinucleotide repeats being predominant (72%), followed by trinucleotide repeats (25%). These markers were validated by designing primer pairs. 25 of these designed primers were tested on a group of 48 apple accessions. All the markers gave robust amplification, generating 93% polymorphism. These findings indicate the usefulness of EST-SSRs in genome analysis. This study further emphasizes the importance of assembly of the vast amounts of data submitted in public databases. Our results have generated a set of non redundant apple ESTs which is of prime importance for development of marker systems without any repetition or overlapping

Regeneration of plantlets through protocorm-like bodies: An alternative method for vanilla micro propagation

388-395An efficient protocol for direct plant regeneration inclusive of rooting and genetic fidelity testing via protocorm-like body (PLB) induction on nodal segments of Vanilla planifolia is presented. Effects of 16 different combinations of plant growth regulators, namely, benzyl aminopurine (BAP), zeatin and naphthalene acetic acid (NAA) were evaluated for PLB induction. BAP at a concentration of 1.5 mg/l together with 2 mg/l NAA in Murashige and Skoog (MS) medium was the most effective in inducing PLBs. Induced PLBs showed good proliferation on peptone supplemented media. Histological observation revealed the sub dermal origin of PLBs, gradually forming shoot apical meristem, shoot primordia and leaf primordia. Mature PLBs regenerated to form shoots on 20 media combinations tested for plantlet regeneration. Best induction rate of 5.08 shoots per PLB after eight weeks of culture was observed on MS medium containing 1 mg/l BAP and 1.5 mg/l NAA. NAA had a stimulatory effect on in vitro rooting with best response recorded on MS medium supplemented with 1 mg/l NAA. Genetic fidelity analysis of well-developed plantlets using inter sequence simple repeat (ISSR) markers revealed no change amongst the tested plants and their mother stocks. Direct regeneration of plantlets by induction of PLBs is an efficient alternative to routine micro-propagation via shoot tips/nodal explants for mass multiplication and germplasm conservation of this commercially important orchid species

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Not AvailableBunium persicum (Boiss), B. Fedtsch or Black cumin, is an economically important medicinal spice that is commonly used as a flavour enhancer and preservative in different food systems. It has also been used in Unani, Iranian, and Indian systems of traditional medicine. A member of the Apiaceae family, it possesses myriad phytochemicals, mainly cuminaldehyde, α-terpinene-7-al, γ-terpinene-7-al, γ-terpinene, ρ-cymene, β-Pinene, etc. and endows various proven therapeutic properties including antioxidant, antimicrobial, anti-inflammatory, lipid/glucose lowering activity, anticarcinogenic activities, etc. This plant grows in the wild in specific areas and is scarcely available and over-exploited; hence, its conservation (both, in vitro and in situ) is a major concern. Besides, negligible work has been done for molecular characterisation, identification and development of promising high yielding cultivars/varieties of this valuable plant. With the aim to attract the attention of potential stakeholders towards the immense potential and infinite qualities of black cumin, this review provides an insight in to the phytochemistry, economic importance, including food and therapeutic uses; morphological, biochemical, and molecular characteristics of Bunium persicum, along with the efforts towards its conservation and a way forward.Not Availabl

Changes in patterns of the double burden of undernutrition and overnutrition in Nepal

This systematic review examined the shifts in undernutrition and overnutrition in Nepal during the past two decades. We searched PubMed for studies and reports published between January 1, 2000, and June 30, 2018. Publications with a sample size greater than or equal to 500 that reported prevalence of nutritional status were included. Six large national reports and 36 studies met study inclusion criteria and were included. Overall, available nationally representative data remained limited. The Nepal Demographic and Health Survey 2001 to 2016 showed that underweight prevalence decreased from 26.7% to 17.2% and prevalence of overweight/obesity increased from 6.5% to 22.1% among women of reproductive age (15-49 years). In preschool children, prevalence of stunting, wasting, and underweight decreased from 57.2% to 35.8%, 11.2% to 9.7%, and 42.7% to 27.0%, respectively. Prevalence of overweight/obesity was low among children and was higher in higher socio-economic status (SES) groups. The overweight-obesity/underweight ratios indicate a shift from undernutrition to overnutrition problem; it was more evident in urban areas and higher SES groups. In conclusion, Nepal is experiencing a nutrition transition. More research is warranted to address this shift, and well-tailored public health efforts need to combat the double burden of overweight/obesity and undernutrition

Silencing of <i>HaAce1</i> gene by host-delivered artificial microRNA disrupts growth and development of <i>Helicoverpa armigera</i>

<div><p>The polyphagous insect-pest, <i>Helicoverpa armigera</i>, is a serious threat to a number of economically important crops. Chemical application and/or cultivation of <i>Bt</i> transgenic crops are the two strategies available now for insect-pest management. However, environmental pollution and long-term sustainability are major concerns against these two options. RNAi is now considered as a promising technology to complement <i>Bt</i> to tackle insect-pests menace. In this study, we report host-delivered silencing of <i>HaAce1</i> gene, encoding the predominant isoform of <i>H</i>. <i>armigera</i> acetylcholinesterase, by an artificial microRNA, <i>HaAce1</i>-amiR1. Arabidopsis pre-miRNA164b was modified by replacing miR164b/miR164b* sequences with <i>HaAce1</i>-amiR1/<i>HaAce1</i>-amiR1* sequences. The recombinant <i>HaAce1</i>-preamiRNA1 was put under the control of CaMV 35S promoter and NOS terminator of plant binary vector pBI121, and the resultant vector cassette was used for tobacco transformation. Two transgenic tobacco lines expressing <i>HaAce1</i>-amiR1 was used for detached leaf insect feeding bioassays. Larval mortality of 25% and adult deformity of 20% were observed in transgenic treated insect group over that control tobacco treated insect group. The reduction in the steady-state level of <i>HaAce1</i> mRNA was 70–80% in the defective adults compared to control. Our results demonstrate promise for host-delivered amiRNA-mediated silencing of <i>HaAce1</i> gene for <i>H</i>. <i>armigera</i> management.</p></div

Designing and construction of pBI::HAR1.

<p>Primer extension PCR was done with a pair of overlapping primers, <i>HAR-</i>F & <i>HAR</i>-R (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0194150#pone.0194150.s006" target="_blank">S1 Table</a>) containing <i>Xba</i>I and <i>Sac</i>I recognition sequences at their 5′ ends. <i>Xba</i>I and <i>Sac</i>I digested PCR product was ligated onto pUC19. pBI121 backbone was generated by digestion with <i>Xba</i>I and <i>Sac</i>I that released <i>GUS</i> coding sequence. After sequence confirmation, <i>HaAce1</i>-preamiRNA1 DNA fragment was released from recombinant pUC19 and ligated onto pBI121 backbone to get pBI::HAR1.</p

Expression of <i>HaAce1</i>-amiR1 in transgenic tobacco lines and down regulation of <i>HaAce1</i> in <i>H</i>. <i>armigera</i> adults.

<p>(A) Northern blot showing <i>HaAce1</i>-amiR1 expression in the transgenic tobacco lines, 17R1 and 29R1. Small RNAs were hybridized with DIG end labelled Anti-amiR<i>Ace</i> probe of 21 nt. C, pBI121 transformed tobacco (vector control); 29R1 & 17R1, <i>HaAce1</i>-amiR1 expression in the selected two transgenic tobacco line, 29R1 and 17R1, respectively; P, labelled Anti-amiR<i>Ace</i> probe. (B) Relative expression of <i>HaAce1</i> in the <i>H</i>. <i>armigera</i> adults. Vector control, <i>HaAce1</i> transcript abundance in <i>H</i>. <i>armigera</i> adults emerged from vector control tobacco treated group; 17R1 and 29R1, <i>HaAce1</i> transcript abundance in deformed <i>H</i>. <i>armigera</i> adults emerged from 17R1 and 29R1 transgenic tobacco treatment groups, respectively. <i>β</i>-<i>Actin</i> gene of <i>H</i>. <i>armigera</i> was used as an internal control. Real time PCR data was analysed using delta-delta Ct method. One way ANOVA test was used to perform the statistical analysis of the data. *** Extremely significant at P<0.001. The test was performed three times.</p

Mortality and developmental deformity in <i>H</i>. <i>armigera</i>.

<p>Thirty second instar larvae of <i>H</i>. <i>armigera</i> were fed continuously on each transgenic line (Line 17R1 and 29R1) and vector control separately (one larva per plate) till their active feeding stage<b>.</b> (A) Mortality percentage of <i>H</i>. <i>armigera</i> larvae. Vector control, mortality percentage in larvae fed on pBI121 transformed tobacco leaves; 17R1, mortality percentage in larvae fed on 17R1 transgenic tobacco line; 29R1, mortality percentage in larvae fed on 29R1 transgenic tobacco line. (B) Deformity percentage of <i>H</i>. <i>armigera</i> adults. Vector control, percentage of emergence of deformed adults from larvae fed on pBI121 transformed tobacco leaves; 17R1, percentage of emergence of deformed adults from larvae fed on 17R1 transgenic tobacco line; 29R1, percentage of emergence of deformed adults from larvae fed on 29R1 transgenic tobacco line. (C) Phenotype of <i>H</i>. <i>armigera</i> adults. Upper panel: Normal adults developed from vector control fed larvae; Lower panel: Deformed adults developed from transgenic fed larvae. One way ANOVA test was used to perform statistical analysis of the data. *** denotes extremely significant differences at P<0.001. The test was repeated three times.</p