Search results for: Dipika Parajulee

Authors: Sarwan Dhir, Suma Basak, Dipika Parajulee

Abstract:

Alfalfa is renowned for its nutritional and biopharmaceutical value as a perennial forage legume. However, establishing a rapid plant regeneration protocol using somatic embryogenesis and efficient transformation frequency are the crucial prerequisites for gene editing in alfalfa. This study was undertaken to establish and improve the protocol for somatic embryogenesis and subsequent plant regeneration. The experiments were conducted in response to natural sensitivity using various antibiotics such as cefotaxime, carbenicillin, gentamycin, hygromycin, and kanamycin. Using 3-week-old leaf tissue, somatic embryogenesis was initiated on Gamborg’s B5 basal (B5H) medium supplemented with 3% maltose, 0.9µM Kinetin, and 4.5µM 2,4-D. Embryogenic callus (EC) obtained from the B5H medium exhibited a high rate of somatic embryo formation (97.9%) after 3 weeks when the cultures were placed in the dark. Different developmental stages of somatic embryos and cotyledonary stages were then transferred to Murashige and Skoog’s (MS) basal medium under light, resulting in a 94% regeneration rate of plantlets. Our results indicate that leaf segments can grow (tolerate) up to 450 mg/L of cefotaxime and 400 mg/L of carbenicillin in the culture medium. However, the survival threshold for hygromycin at 12.5 mg/L, kanamycin at 250 mg/L, gentamycin at 50 mg/L, and timentin (300 mg/L). The experiment to improve the protocol for achieving efficient transient gene expression in alfalfa through genetic transformation with the Agrobacterium tumefaciens pCAMBIA1304 vector was also conducted. The vector contains two reporter genes such as β-glucuronidase (GUS) and green fluorescent protein (GFP), along with a selectable hygromycin B phosphotransferase gene (HPT), all driven under the CaMV 35s promoter. Various transformation parameters were optimized using 3-week-old in vitro-grown plantlets. The different parameters such as types of explant, leaf ages, preculture days, segment sizes, wounding types, bacterial concentrations, infection periods, co-cultivation periods, different concentrations of acetosyringone, silver nitrate, and calcium chloride were optimized for transient gene expression. The transient gene expression was confirmed via histochemical GUS and GFP visualization under fluorescent microscopy. The data were analyzed based on the semi-quantitative observation of the percentage and number of blue GUS spots on different days of agro-infection. The highest percentage of GUS positivity (76.2%) was observed in 3-week-old leaf segments wounded using a scalpel blade of 11 size- after 3 days of post-incubation at a bacterial concentration of 0.6, with 2 days of preculture, 30 min of bacterial-leaf segment co-cultivation, with the addition of 150 µM acetosyringone, 4 mM calcium chloride, and 75 µM silver nitrate. Our results suggest that various factors influence T-DNA delivery in the Agrobacterium-mediated transformation of alfalfa. The stable gene expression in the putative transgenic tissue was confirmed using PCR amplification of both marker genes, indicating that gene expression in explants was not solely due to Agrobacterium, but also from transformed cells. The improved protocol could be used for generating transgenic alfalfa plants using genome editing techniques such as CRISPR/Cas9.

Keywords: Medicago sativa l. (Alfalfa), agrobacterium tumefaciens, β-glucuronidase, green fluorescent protein, transient gene

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Authors: Tahammal Hussain, Khuram Zia, Mumammad Jalal Arif, Megha Parajulee, Abdul Hakeem

Abstract:

The Bioassay on neonate, 2nd and 3rd instar larvae of Helicoverpa armigera (Hubner) were conducted against Bacillus thuringiensis proteins Cry1Ac. Cry1Ac was incorporated into an artificial diet and was serially diluted with distilled water and then mixed with diet at an appropriate temperature of diet. Toxins incorporated prepared diet was poured into Petri-dishes. For controls, distilled water was mixed with the diet. Five toxin doses 0.25, 0.5, 1, 2, and 4 ug / ml and one control were used for each instars of H. armigera 20 larvae were used in each replication and each treatment is replicated four times. LC50 of Cry1Ac against neonate, 2nd and 3rd instar larvae of H. armigera were 0.34, 0.81 and 1.46 ug / ml. So Cry1Ac is more effective against neonate larvae of H .armigera as compared to 2nd and 3rd instar larvae under laboratory conditions.

Keywords: B. thuringiensis, Cry1Ac, H. armigera, toxicity

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Authors: Saranya Ganapathy, Megha N. Parajulee, Michael San Francisco, Hong Zhang

Abstract:

Insect pests are a serious threat to agricultural productivity. Use of chemical pesticides, the predominant control method thus far, has resulted in environmental damage, pest resurgence, and negative effects on non-target species. Genetically modified (GM) crops offer a promising alternative, and Bacillus thuringiensis endotoxin genes have played a major role in this respect. However, to overcome insect tolerance issues and to broaden the target range, it is critical to identify alternative-insecticidal toxins working through novel mechanisms. Our research group has identified a kinase from Chilo iridescent virus (CIV; Family Iridoviridae) that has insecticidal activity and designated it as ISTK (Iridovirus Serine/Threonine Kinase). A 35 kDa truncated form of ISTK, designated iridoptin, was obtained during expression and purification of ISTK in the yeast system. This yeast-expressed CIV toxin induced 50% mortality in cotton aphids and 100% mortality in green peach aphids (GPA). Optimized viral genes (o-ISTK and o-IRI) were stably transformed into the model plant, Arabidopsis. PCR analysis of genomic DNA confirmed the presence of the gene insert (oISTK/oIRI) in selected transgenic lines. The further screening was performed to identify the PCR positive lines that showed expression of respective toxins at the polypeptide level using Western blot analysis. The stable lines expressing either of these two toxins induced moderate to very high mortality in GPAs and significantly affected GPA development and fecundity. The aphicidal potential of these transgenic Arabidopsis lines will be presented.

Keywords: Chilo iridescent virus, insecticidal toxin, iridoviruses, plant-incorporated protectants, serine/threonine kinase

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Authors: Kavita Khoiwal, Vatsla Dadhwal, K. Aparna Sharma, Dipika Deka, Plabani Sarkar

Abstract:

Introduction: About 2.4 million (1.93 - 3.04 million) people are living with HIV/AIDS in India. Of all HIV infections, 39% (9,30,000) are among women. 5.4% of infections are from mother to child transmission (MTCT), 25,000 infected children are born every year. Besides the risk of mother to child transmission of HIV, these women are at risk of the higher adverse pregnancy outcome. The objectives of the study were to compare the obstetric and neonatal outcome in women who are HIV positive with low-risk HIV negative women and effect of antiretroviral drugs on preterm birth and IUGR. Materials and Methods: This is a retrospective case record analysis of 212 HIV-positive women delivering between 2002 to 2015, in a tertiary health care centre which was compared with 238 HIV-negative controls. Women who underwent medical termination of pregnancy and abortion were excluded from the study. Obstetric outcome analyzed were pregnancy induced hypertension, HIV positive intrauterine growth restriction, preterm birth, anemia, gestational diabetes and intrahepatic cholestasis of pregnancy. Neonatal outcome analysed were birth weight, apgar score, NICU admission and perinatal transmission.HIV-positiveOut of 212 women, 204 received antiretroviral therapy (ART) to prevent MTCT, 27 women received single dose nevirapine (sdNVP) or sdNVP tailed with 7 days of zidovudine and lamivudine (ZDV + 3TC), 15 received ZDV, 82 women received duovir and 80 women received triple drug therapy depending upon the time period of presentation. Results: Mean age of 212 HIV positive women was 25.72+3.6 years, 101 women (47.6 %) were primigravida. HIV positive status was diagnosed during pregnancy in 200 women while 12 women were diagnosed prior to conception. Among 212 HIV positive women, 20 (9.4 %) women had preterm delivery (< 37 weeks), 194 women (91.5 %) delivered by cesarean section and 18 women (8.5 %) delivered vaginally. 178 neonates (83.9 %) received exclusive top feeding and 34 neonates (16.03 %) received exclusive breast feeding. When compared to low risk HIV negative women (n=238), HIV positive women were more likely to deliver preterm (OR 1.27), have anemia (OR 1.39) and intrauterine growth restriction (OR 2.07). Incidence of pregnancy induced hypertension, diabetes mellitus and ICP was not increased. Mean birth weight was significantly lower in HIV positive women (2593.60+499 gm) when compared to HIV negative women (2919+459 gm). Complete follow up is available for 148 neonates till date, rest are under evaluation. Out of these 7 neonates found to have HIV positive status. Risk of preterm birth (P value = 0.039) and IUGR (P value = 0.739) was higher in HIV positive women who did not receive any ART during pregnancy than women who received ART. Conclusion: HIV positive pregnant women are at increased risk of adverse pregnancy outcome. Multidisciplinary team approach and use of highly active antiretroviral therapy can optimize the maternal and perinatal outcome.

Keywords: antiretroviral therapy, HIV infection, IUGR, preterm birth

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Authors: C. Shekhar Parajulee

Abstract:

Nepal practiced a centralized unitary governing system for a long and has gone through the federal system after the promulgation of the new constitution on 20 September 2015. There is a big paradigm shift in terms of governance after it. Now, there are three levels of governments, one federal government in the center, seven provincial governments and 753 local governments. Federalism refers to a political governing system with multiple tiers of government working together with coordination. It is preferred for self and shared rule. Though it has opened the door for rights of the people, political stability, state restructuring, and sustainable peace and development, there are many prospects and challenges for its proper implementation. This research analyzes the discourses of federalism implementation in Nepal with special reference to one of seven provinces, Gandaki. Federalism is a new phenomenon in Nepali politics and informed debates on it are required for its right evolution. This research will add value in this regard. Moreover, tracking its evolution and the exploration of the attitudes and behaviors of key actors and stakeholders in a new experiment of a new governing system is also important. The administrative and political system of Gandaki province in terms of service delivery and development will critically be examined. Besides demonstrating the performances of the provincial government and assembly, it will analyze the inter-governmental relation of Gandaki with the other two tiers of government. For this research, people from provincial and local governments (elected representatives and government employees), provincial assembly members, academicians, civil society leaders and journalists are being interviewed. The interview findings will be analyzed by supplementing with published documents. Just going into the federal structure is not the solution. As in the case of other provincial governments, Gandaki had also to start from scratch. It gradually took a shape of government and has been functioning sluggishly. The provincial government has many challenges ahead, which has badly hindered its plans and actions. Additionally, fundamental laws, infrastructures and human resources are found to be insufficient at the sub-national level. Lack of clarity in the jurisdiction is another main challenge. The Nepali Constitution assumes cooperation, coexistence and coordination as the fundamental principles of federalism which, unfortunately, appear to be lacking among the three tiers of government despite their efforts. Though the devolution of power to sub-national governments is essential for the successful implementation of federalism, it has apparently been delayed due to the centralized mentality of bureaucracy as well as a political leader. This research will highlight the reasons for the delay in the implementation of federalism. There might be multiple underlying reasons for the slow pace of implementation of federalism and identifying them is very tough. Moreover, the federal spirit is found to be absent in the main players of today's political system, which is a big irony. So, there are some doubts about whether the federal system in Nepal is just a keepsake or a substantive.

Keywords: federalism, inter-governmental relations, Nepal, provincial government

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