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Strategies for Enhanced Production of Commercially Important Secondary Metabolites from in Vitro Established Cultures of Linum Usitatissimum L.

Thesis Info

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Author

Zahir, Adnan

Program

PhD

Institute

Quaid-I-Azam University

City

Islamabad

Province

Islamabad.

Country

Pakistan

Thesis Completing Year

2019

Thesis Completion Status

Completed

Subject

Biotechnology

Language

English

Link

http://prr.hec.gov.pk/jspui/bitstream/123456789/11886/1/Adnan%20zahir%20biotech%202019%20QAU%20prr.pdf

Added

2021-02-17 19:49:13

Modified

2024-03-24 20:25:49

ARI ID

1676727242333

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Linum usitatissimum is an important cultivated, commercial and medicinal plant in the family Linacaea. This plant is also known as flax and their seeds are famous for their nutritional, medicinal and industrial purposes. Exploitation of Linum usitatissimum is context dependent. Commercially, it is used for manufacturing of linen fiber hence the name flaxseed is given to such a variety of Linum usitatissimum, on the other hand seeds of a variety used for many nutritional and industrial applications are called linseed. Linum usitatissimum is generally cultivated in areas having moderate to cold climate. The plant has a slender stem, lanceolate leaves and pale blue flowers with five petals. Canada, Russia, United States, China and India are among the top growers of Linum usitatissimum. Worldwide production of flax is 2.5-3 million tonnes per year. This plant is rich in pharmaceutical compounds along with some important nutraceuticals. Novel compounds found in flax are polyphenols, comprising of two major classes—lignans and neolignans. Lignans like secoisolariciresinol diglucoside (SDG) and lariciresinol diglucoside (LDG) are potential anticancer compounds whereas, neolignans such as dehydrodiconiferyl alcohol glucoside (DCG) and guaiacylglycerol-β-coniferyl alcohol ether glucoside (GGCG) are used in antimicrobial and anti-inflammatory drugs. However, the yield of these medicinal compounds in natural habitat is low and their extraction from wild is an expensive process. For this reason, researchers are busy devising cost effective, reproducible and practical strategies to enhance producton of lignans and neolignans in in vitro established infrastructure. In the first experiment, we established callus cultures of Linum usitatissimum following protocol of Anjum et al. (2016). These cultures were derived from stem explants obtained from in vitro seed derived aseptic plantlets. The objective of this study was to evaluate the effects of qualitative and quantitative differences in culture medium along with varying photoperiod treatments, on growth kinetics and secondary metabolites production in callus cultures of Linum usitatissimum. To this end, ~1 g callus was inoculated on three different culture media, namely Murashige and Skoog, Gamborg B5 and Schenk and Hildebrandt, and each culture on specific growth medium was kept under the influence of different photoperiod treatments viz. 16/8 h light and dark, continuous light and continuous dark, respectively. We observed differential effects of nutrient and photoperiods variation on growth kinetics and secondary metabolites xiii accumulation in callus cultures of Linum usitatissimum. Significant growth rate was observed on Gamborg B5 medium as compared to Murashige and Skoog medium, while Schenk and Hildebrandt medium showed a slow but steady growth response. Similarly, we observed that Gamborg B5 medium enhanced callus biomass (fresh weight 413 g/l anddry weight 20.7 g/l), phenolics production (667.60 mg/l), and lignan content (secoisolariciresinol diglucoside6.33 andlariciresinol diglucoside 5.22 mg/gdry weight respectively) at 16/8 h light and dark-week 4, while that of neolignans (dehydrodiconiferyl alcohol glucoside 44.42 and guaiacylglycerol-β-coniferyl alcohol ether glucoside 9.26 mg/g dry weight, respectively) at continuous dark-week 4. Conversely, maximum flavonoid production occurred at both Murashige and Skoog, Schenk and Hildebrandt media in the presence of continuous light. Generally, continuous dark had no significant role in any growth associated parameter. In the second experiment, we established cell suspension cultures of Linum usitatissimum by modifying method of Anjum et al. Simply, ~3g callus was inoculated into 100 ml liquid Murashige and Skoog medium and kept on a shaking incubator for 15 days in controlled conditions. Ten milliliters media containing viable cells was used as inoculum for establishment of cell suspension cultures. This experiment was aimed to enhance growth associated parameters and secondary metabolites accumulation with the help of repeated introduction of chemogenic silver nanoparticles at different stages, into cell suspension cultures of Linum usitatissimum. Repeated addition of silver nanoparticles enhanced biomass and polyphenols accumulation in cell suspension cultures. Adding silver nanoparticles on day 10 resulted in comparatively, highest production of lignans (secoisolariciresinol diglucoside, 252.75 mg/l; lariciresinol diglucoside, 70.70 mg/l), neolignans (dehydrodiconiferyl alcohol glucoside, 248.20 mg/l; guaiacylglycerol-β-coniferyl alcohol ether glucoside, 34.76 mg/l), total phenolic content (23.45 mg GAE/g DW), total flavonoid content (11.85 mg QUE/g DW) and biomass (dry weight: 14.5 g/l), respectively. Optimum production of both lignans and neolignans occurred on day 20 of culture; a 10-fold increase in secoisolariciresinol diglucoside, 2.8 fold increase in lariciresinol diglucoside, 5 fold increase in dehydrodiconiferyl alcohol glucoside and 1.75-fold increase in guaiacylglycerol-β-coniferyl alcohol ether glucoside was observed in production levels compared to control treatments, respectively. xiv In the third experiment, cell suspension cultures of Linum usitatissimum were exposed to repeated addition of biogenic zinc oxide nanoparticles in order to evaluate its effect on biomass accumulation and secondary metabolites production. Zinc oxide nanoparticles were added at three different stages and their results were compared to control treatments. Repeated elicitation of cell suspension cultures on day 0 and 15 resulted in highest fresh weight (412.16 g/l) and lignans production (secoisolariciresinol diglucoside 284.12 mg/l: lariciresinol diglucoside 86.97 mg/l). Contrarily, repeated elicitation on day 0 and 25 resulted in highest dry biomass (13.53 g/l), total phenolic production (537.44 mg/l), total flavonoid production (123.83 mg/l) and neolignans production (dehydrodiconiferyl alcohol glucoside 493.28 mg/l: guaiacylglycerol-β-coniferyl alcohol ether glucoside 307.69 mg/l). Enhancement of plant growth, free radical scavenging capacity and secondary metabolites accumulation was several fold greater than control treatments.
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سیاسی اضطرابی صورتحال کا جائزہ اور اس کا حل: سیرۃ نبویﷺ کی روشنی میں

Pakistan is an Ideological country, there are only two ideological states in the world. First one was Madinah (Saudi Arabia) and the second one is Pakistan. Political situation in our country is worst. There is no any stability in political situation. From its existence Pakistan is facing different problems and issues. In this research article, will try to draw reasons and will table solutions to get political stability in Pakistan.

Parasitic Potential of Encarsia Sophia Girault and Dodd on Bemisia Tabaci Gennadius Reared on Different Host Plants

Study was conducted at Insectary Biological Control Labs., IPMP, National Agricultural Research Centre (NARC) Islamabad under controlled environmental conditions at 26±1°C, 60±5% R.H. and 12:12 L: D photoperiod. Research experiments were done on whitefly Bemisia tabaci (Gennadius) which has been a serious pest in Pakistan for over the past two decades and its potential parasitoid Encarsia sophia (Girault & Dodd) as it is invasive, can be mass reared easily and able to parasitize more hosts. Results regarding host plant effect (cotton, brinjal and tomato) on B. tabaci biology concluded that development times, ovipositional preferences and survival of immature stages was strongly mediated by host plants selected for rearing. Among all the selected host plants, brinjal was considered as most suitable host in terms of developmental times (19.95±0.22 days) as compare to tomato (20.77±0.24 days) and cotton (23.05±0.23 days). Mean population density of eggs on cotton showed stronger preferences of B. tabaci for oviposition among three host plants with a mean number of (194.20±7.69) eggs laid/female with a mortality rate of (23.33%). According to the results, host plants selected for rearing of B. tabaci also showed significant effect on the biology of E. sophia. E. sophia developmental times (egg-adult) in B. tabaci instars was significantly shorter for parasitoids that emerged from brinjal (13.50±0.16 days) with highest percentage of parasitism (29.00±2.12) ranged from 15.00-55.00 and calculated higher emergence on brinjal 92.60±1.71% over other host plants. Results on population growth parameters also exposed brinjal as a good and effective host choice as parasitoid populations develops successfully on it with significantly higher net reproductive rate (Ro) 22.83 andintrinsic rate of natural increase (rm) 0.1043 with shorter mean generation time (Tc) 29.99 and doubling time (DT) 6.64 as compared to reared on cotton and tomato. The immature developmental stages observed for E. sophia were as eggs, 1st, 2nd and 3rd larval instars, pre-pupa and black pupae. Mean development duration from egg deposition till adult emergence was significantly different 12.61±0.13 days for female and 13.94±0.16 days for male. E. sophia preferred to oviposit in all four host instars of B. tabaci although preference differences were observed among host stages in terms of parasitism and feeding behaviours. Parasitism was observed higher on 3rd and 4th instars both in No-choice and Multiple- instar choice experiments but feeding was relatively higher in younger instars. Emergence was found both in light and dark conditions suggesting activity of parasitoid throughout 24h. Newly emerged parasitoid when placed under light source created ease for mating. Honey significantly prolonged the longevity of E. sophia adult. E. sophia fitness and efficacy in terms of fecundity, parasitism, developmental time, pupal survival and longevity was significantly altered by host instars. Almost all biological attributes of E. sophia were at its best developed on 3rd instars of B. tabaci with a faster developmental time (11.80±0.20 days), emerged more synchronously (86±2.66%) and lived longer (12.95±0.78 days) with maximum oviposition rate (62.50±1.69) eggs/female. Therefore it was concluded that larger hosts tended to produce better parasitoids than smaller hosts. The functional response studies determined that E. sophia have the ability to adjust itself at higher densities. E. sophia in relation to the density of host fitted the description of a type II functional response. Results commended a density of 30 instars as the highest critical density for parasitoid where parasitism rate and number of progeny was maximum with a minimum rate of super-parasitism. Host discrimination behaviour of E. sophia was also observed and it was concluded that newly emerged females NEF exhibit less ability to discriminate between the parasitised and unparasitised hosts over SHF and DEF. Females used to exploit minimum hosts when offered an unparasitized host while exploited maximum hosts when offered with a parasitized host as. Mean handling time for E. sophia was higher on unparasitized host while minimum for already parasitized host. The information will be useful in designing mass rearing protocols and in release trials of E. sophia for suppression of B. tabaci populations.