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Home > Ri-Mediated Genetic Transformation in Licorice Glycyrrhiza Uralensis and Lycium Species by Establishing Hairy Root Cultures.

Ri-Mediated Genetic Transformation in Licorice Glycyrrhiza Uralensis and Lycium Species by Establishing Hairy Root Cultures.

Thesis Info

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Author

Chahel, Ayesha Arif

Program

PhD

Institute

Lahore College for Women University

City

Lahore

Province

Punjab

Country

Pakistan

Thesis Completing Year

2019

Thesis Completion Status

Completed

Subject

Botany

Language

English

Link

http://prr.hec.gov.pk/jspui/bitstream/123456789/10792/1/Final%20thesis%2022-09-2018.docx

Added

2021-02-17 19:49:13

Modified

2024-03-24 20:25:49

ARI ID

1676727105455

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Glycyrrhiza uralensis Fisch, Lycium ruthenicum Murr. and L. barbarum L. are important medicinal plant species. These plants have various biologically active compounds such as phenolics, alkaloids, anthocyanins and flavonoids. These secondary metabolites are being used extensively in dietary food and pharmaceutical products. Genes ERF061 and TCP4 belong to transcription factors family ERF and TCP. However these two genes ERF061 and TCP4 are well studied in tomato (fruit ripening) and model plant Arabidopsis (Plant organ morphogenesis). Still function of these transcription factors in plant secondary metabolites production in model and non-model plant species are not well understood. Identification and characterization of unknown metabolites under Agrobacterium rhizogenes stress and effect of selected transcription factors in comparative plant species lead to novel discoveries in non-model plant species. Comparative metabolomic analyses of two plant species from same genus (Lycium species) and one species from different genus (G. uralensis with well documented metabolites analysis) probably provide more accurate gene function predictions. Therefore for the first time present study was proposed to induce hairy root culture system in G. uralensis, L. ruthenicum Murr. and L. barbarum with over-expression vectors of LrERF061 and LrTCP4 to predict their most possible function in achieving high yield of phenolic polyamines and other non-targeted secondary metabolites. Since, phenolics are medicinally significant compounds present in roots at very low concentration and it is expensive to produce them commercially. Over-expression sequence of ERF061 and TCP4 gene was retrieved from the transcriptome data of L. ruthenicum (LrERF061-OE and LrTCP4-OE) and transferred to genome of selected plant species by Ri-mediated genetic transformation. The successful incorporation of LrERF061-OE and LrTCP4-OE into genome of putative transgenic hairy root clones was confirmed by GUS and Polymerase Chain Reaction (PCR) analyses. Three different A. rhizogenes strains ARqua-1, MSU440 and R1000 were evaluated for maximum hairy root production. A. rhizogenes strain R1000 proved best strain with hypocotyl showing maximum transformation efficiencies 80% and 44% in L. ruthenicum and L. barbarum respectively. While A. rhizogenes strain ARqua-1 proved best with transformation efficiency (60%) in hypocotyls of G. uralensis. Two different explants (leaf and hypocotyls) were also tested for high production of hairy root in G. uralensis, L. ruthenicum and L. barbarum. In present investigation best explant was hypocotyl for generating healthy and rapidly growing transgenic hairy root clones (THRC). Transgenic and non-transgenic hairy roots of L. ruthenicum and L. barbarum were rapidly growing on MS medium with 1X B5 vitamins supplemented with 3% sucrose. In contrast THRC of G. uralensis showed best growth on B5 medium with 1X B5 vitamins having 3% sucrose. Effect of various hygromycin concentrations were also evaluated on THRC of tested plant species. The optimum concentration of hygromycin for growth of THRC in G. uralensis, L. ruthenicum and L. barbarum was 10mg/L. Real-time quantitative PCR (qRT-PCR) results suggested that the transcripts of LrERF061-OE and LrTCP4-OE were dominant almost 2 times in L. ruthenicum and LrERF061-OE in L. barbarum hairy root samples compared to control with different transcription levels. Transgenic callus was also established from THRC of L. ruthenicum and L. barbarum. Transgenic callus of both L. ruthenicum and L. barbarum appeared more green and massive in growth compared to non-transgenic callus. Non-transgenic callus was light yellow and slightly brown in color and later died on culture media. Transgenic and non-transgenic hairy root clones with high growth rate cultured in liquid MS medium to assure enough hairy root samples for metabolites analyses. Transgenic hairy root clones of G. uralensis were unhealthy and not good in growth. Therefore G. uralensis hairy roots clones were not subjected to metabolites analysis due to less hairy root samples availability. Various un-known and novel root specific metabolites of L. ruthencium and L. barbarum were tentatively identified by using ultrahigh performance liquid chromatography coupled to photodiode array detector/ quadrupole time-of-flight mass spectrometry (UPLC-PDA-qTOF-MS). Through this non-targeted metabolomics function of LrERF061-OE and LrTCP4-OE was studied in non model plant species (L. ruthenicum and L. barbarum). Interestingly transgenic hairy root clones of L. ruthenicum with LrTCP4-OE and LrERF061-OE showed high relative abundance of kukoamine A and 34 other secondary metabolites compared to control type hairy roots. In THRC of 06 metabolites showed maximum fold change >1000. While 06 metabolites in THRC of L. ruthencium with LrTCP4-OE showed maximum fold change >200. L. barbarum with LrERF061-OE produced total 24 metabolites. From these 24 metabolites, 21 metabolites showed high relative abundance in THRC compared to control hairy root clones. Total 13 metabolites showed maximum fold change >10 in these THRC of L. barbarum with LrERF061-OE. While trials using LrTCP4-OE proved unsuccessful in L. barbarum hairy root production.After growth period of 1 month, the best growing lines of L. ruthenicum with LrTCP4-OE showed 0.14% kukoamine A level more than control hairy roots 0.11% compared with authentic standard in UPLC analysis. The enhanced productivity correlated to increased LrTCP4-OE activity, validating primary role that LrTCP4-OE played for total kukoamine A synthesis and efficiency of the non-targeted techniques of metabolomics in studying plant metabolites. The protocol of genetic transformation used in this work can be used in other plant species to study target gene function in secondary metabolites production and regulation. Further role of LrTCP4-OE was also confirmed in phenolic polyamine biosynthesis (particularly kukoamine A) in phenylpropanoid biosynthetic pathway.
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ملا طاہر سیف الدین

ملا طاہر سیف الدین
گذشتہ دو مہینوں میں مسلمانوں کے دو بڑے قومی حادثے ہوئے، ۵؍ نومبر کو داؤدی بوہرون کے امام ملا طاہر سیف الدین نے انتقال کیا، ان کی ذات جامع صفات تھی، بڑے ذی علم، دیندار، فیاض و مخیر اور وسیع القلب تھے، دینی علوم پر ان کی نگاہ بہت وسیع تھی، اس لحاظ سے وہ ہندوستان کے ممتاز علماء میں تھے، صاحبِ قلم بھی تھے، عربی میں ان کی کئی تصانیف ہیں، انھوں نے اپنے دور میں نہ صرف اپنے فرقہ کی بڑی تعلیمی و اقتصادی خدمت کی بلکہ دوسرے اسلامی فرقوں کے ساتھ بھی ان کا سلوک روادرانہ و فیاضانہ تھا، اور ان کو ایک دوسرے کے قریب لانے کی کوشش کی، مسلم یونیورسٹی کے تو چانسلر ہی تھے، اس کو وقتاً فوقتاً بڑی بڑی رقمیں دیتے رہتے تھے، دارالمصنفین کی جوبلی کے موقع پر اس کو بارہ ہزار کا عطیہ دیا، اس لیے ہر فرقہ کے مسلمانوں میں عزت و وقعت کی نظر سے دیکھے جاتے تھے، اﷲ تعالیٰ ان کے حسنات کے طفیل میں ان کی مغفرت فرمائے، دارالمصنفین اس حادثہ میں ان کے لائق جانشین ملا برہان الدین کا شریک غم ہے اور دعا ہے کہ خدا ان کو ان کے باعظمت والد کے نقش قدم پر چلنے کی توفیق عطا فرمائے۔ (شاہ معین الدین ندوی، دسمبر ۱۹۶۵ء)

 

Hematological, Biochemical and Etiological factors of Chronic Liver Disease in a tertiary care Hospital of Lahore

Chronic Liver Disease (CLD) progresses from hepatocyte inflammation to fibrosis, regeneration, cirrhosis and in some cases to Hepatocellular Carcinoma (HCC). In general, the main etiologies of Liver Cirrhosis (LC) are viral infections (hepatitis C and B viruses), chronic alcohol abuse and Non-Alcoholic Fatty Liver Disease (NAFLD), including Non-Alcoholic SteatoHepatitis (NASH). Major complications of CLD are ascites, upper gastrointestinal bleeding, jaundice (acute or chronic) and hepatic encephalopathy. Objectives: This study assesses the etiological factors and complications of CLDin a tertiary care hospital of Lahore, Pakistan. Study Design: Cross-sectional. Methods: Study was carried out in indoor and Accident & Emergency Departments of Mayo Hospital Lahore. 100 clinically diagnosed CLD cases were chosen through “Convenient Sampling” technique during 3 months. Observations: Most common complications of CLD were upper GI variceal Bleeding (48%) & hepatic encephalopathy (34%) and acute or chronic hepatitis (AVH) (33%). Other less common complications observed were hepatorenal syndrome (10%), Spontaneous bacterial peritonitis (15%), Ascites (5%) and HCC (10%). Conclusions: Hepatitis C was found as main etiological factor of CLD. Bleeding andhepatic encephalopathy are the common complications. Awareness programmes regarding CLD and its complications are mandatory in our society to improve human health.

Species Diversity of Basidiomycetes of District Malakand

This dissertation communicates the results of a scientific endeavor undertaken for exploring the diversity of Basidiomycetous fungi of District Malakand, Pakistan. The District Malakand is an administrative unit of the Khyber Pakhtunkhwa province, occupies 952 Km2 land within the geographical limits of 34⁰ 35’ to 34⁰ 66’ N and 71⁰ 65’ to 72⁰ 25’ E. Collections of mushrooms, rust and smut fungi were made from different area of Malakand District during the years 2013 and 2014. Field data of the specimens were recorded on the spot, proper photographs were taken and the collection was dried and preserved accordingly. More than 150 specimens of Basidiomycota were collected, comprising of 51 taxa belonging to 17 families. Distribution pattern of the groups shows that Agaricomycotina, the mushroom forming fungi, was the largest group represented by 39 species, Pucciniomycotina, the pathogenic rust fungi by 11 species and the smut fungi, Ustilaginomycotina, with a single species viz., Urocystis avenastri. All these fungi were characterized morpho-anatomically. The members of Agaricomycotina were also subjected to molecular identification, based on four gene regions viz., ITS, nrLSU, rpb2, tef-1α. Phylogenies of the analyzed taxa were elaborated with maximum likelihood, maximum parsimony and Bayesian analysis. Our research outputs includes one new species to science viz., Tulotoma ahmadii. Ten more taxa viz., Agaricus malakandensis nom. prov., A. minorus nom. prov., Cantharocybe pakistanicus nom. prov., Coprinopsis hispidus nom. prov., Hymenagaricus pakistaniensis nom. prov., Leucoagaricus badius nom. prov., La. xiii sultanii nom. prov., Parasola lentiformis nom. prov., Pa. malakandensis nom. prov. and Psathyrella fusiformis nom. prov., are in the process of recognition / publication as new species. Furthermore, other 12 species viz., Agrocybe semiorbicularis, Conocybe papillata, Coprinellus curtus, Marasmiellus palmivorus, Pleurotus cystidiosus, Parasola lilatincta, Pa. schroeterii, Stropharia ambigua, Puccinia microspora, P. nakanishikii, Termitomyces umkowaan and Trametes lactinea are added as new records to the species list of the fungi of Pakistan. In Agaricomycotina, Agaricaceae proved to be the most frequent family represented by six genera (Agaricus, Hymenagaricus, Leucoagaricus, Leucocoprinus, Lycoperdon and Tulostoma). The second largest family was Psathyrellaceae with four genera viz., Coprinellus, Coprinopsis, Parasola and Psathyrella. Only two genera were recorded for each the family i.e., Omphalotaceae, Polyporaceae and Strophariaceae. Whereas families like Bolbitaceae, Hygrophoraceae, Hymenocheataceae, Geastraceae, Lyophyllaceae, Miripelaceae, Mirulaceae, Pleurotaceae and Pluteaceae were represented by a single genus each i.e., Conocybe, Cantharocybe, Phillanus, Geastrum, Termitomyces, Rigidoporus, Irpex, Pleurotus and Volvariella, respectively. Pucciniomycotina was represented by two families, Phragmidiaceae (Phragmidium) and Puccineaceae represented by (Puccinia, Uromyces). The Ustiloginomycotina was monotypic only represented by Urocystis avenastri. This study concluded that low altitude mountains, foothills and grassy lands of Malakand are rich in fungi species and needs exploration of other groups also. These findings have not only upgraded the existing knowledge of basidiomycetes Mycota from District Malakand but also established a sound baseline for future research and exploitation of fungi in Pakistan.