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Biotarnsformation of Terpenes Using Medicinal Labiates and Fusion Effect on the Products

Thesis Info

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Author

Hammad Afzal Kayani

Program

PhD

Institute

University of Karachi

City

Karachi

Province

Sindh

Country

Pakistan

Thesis Completing Year

2015

Thesis Completion Status

Completed

Subject

Biotechnology

Language

English

Link

http://prr.hec.gov.pk/jspui/bitstream/123456789/13468/1/Hammad_Afzal_Kayani_Biotechnology_2015_HSR_UoK_Karachi_02.09.2016.pdf

Added

2021-02-17 19:49:13

Modified

2024-03-24 20:25:49

ARI ID

1676725646452

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The thesis was divided into parts; first part consisted of plant cell and tissue culture studies, while second part constituted biotransformation studies using Labiates cell culture and genetic modification effect on biotransformed products. PART A In first part, in vitro protocol for direct regeneration of Lamiaceae plants were established for biotransformation and other modification studies. For this, three Labiates (Ocimum basilicum, Ocimum sanctum and Agastache anisata) were studied. In O. basilicum, the best response for direct plant regeneration was observed in Murashige and Skoog (MS) culture medium containing IAA 0.025 mg/L + BAP 0.1 mg/L, producing 16.4 shoots having a shoot length of 5.33 cm, and 6.86 roots having a root length of 5.36 cm. The plants were best acclimated in sand: farm yard manure (50:50 w/w) and sand (100 %). In O. sanctum, direct plant regeneration was observed in MS medium containing IAA 0.025 mg/L + BAP 0.1 mg/L, producing 12.4 shoots with a shoot length of 5.94 cm, and inducing 15.0 roots having a root length of 6.05 cm. The plants were best acclimated in sand: farm yard manure (50:50) and sand (100 %). Till date, this is the first report of in vitro propagation of O. basilicum using nodal segment as explants. In Agastache anisata, plant regeneration was observed in MS medium containing IAA 0.05 mg/L + BAP 0.1 mg/L, producing 7.93 shoots with a shoot length of 6.0 cm, and inducing 5.96 roots having a root length of 4.01 cm. The plants were best acclimated in sand: farm yard manure (50:50) and sand (100 %). Till date, this is the first report of in vitro propagation of A. anisata using tissue culture technology. The plant was tested for its bioactivity, and it exhibited potent cytotoxic activity. In order to establish and optimize callus induction protocol, required for the biotransformational studies, different combinations of plant growth regulators (PGR) with MS medium were tested. Out of 60 different combinations, best combination for O. sanctum was DN6 (MS + NAA 2 mg/L + 2,4- D 0.25 mg/L) that produced friable callus. When this callus was transferred into the liquid medium, it started dispersing easily making good suspension culture for biotransformation studies. In O. basilicum, yellow friable granular callus was induced in DN9 (MS + NAA 2 mg/L + 2,4-D 0.5 mg/L), and this was further used for the establishment of cell suspension culture for the biotransformational studies. PART B For the biotransformational studies, six substrates were used in which five were terpenes and one steroid. In total, fifteen compounds were isolated for the first time via this route (plants), but twelve (2, 3, 6, 8-11, 13, 16, 18, 19 and 21) are known and three are new (4, 5 and 14) metabolites. Known compounds were mostly synthetically produced compounds or/and some of them bioconverted from fungus but not through plant cell suspension cultures. These compounds have not been acquired this way by any plant cell culture. In order to seek different derivatives of a given compound; various cell suspension cultures were used against several kind of compounds to get more valuable metabolites. In this connection, podocarpic acid (1) was subjected to biotransformation process employing cell suspension culture of Ocimum species. Both cell culture (Ocimum) and substrate (1) were used for the first with each other. Biotransformation of podocarpic acid (1) by cell suspension culture of Ocimum yielded compounds Methyl podocarpate (2), Podocarpic acid acetate (3), 6α, 7α-epoxy podocarpic acid (4), 6β, 7β-epoxy podocarpic acid (5) and 7-keto podocarpic acid (6). Metabolites 4 and 5 were found to be new metabolites, as deduced on the basis of spectroscopic techniques. Ethynodiol diacetate (7), a steroidal compound has not previously been used for the biotransformation employing the cell suspension culture of Ocimum basilicum. Biotransformation of ethynodiol diacetate (6) by culture of Ocimum basilicum yielded compounds 8-11. 17α-Ethynyl-17β- acetoxyestr-4-en-3-one (8), 17α-Ethynyl-17β-hydroxyestr-4-en-3-one (9), 17α-Ethynyl-3β-hydroxy- 17β-acetoxyestr-4-ene (10) and 17α-Ethynyl-5α,17β-dihydroxyestr-3-ene (11). When totarol (12) was incubated with the cell suspension culture of O. sanctum for 20 days, two metabolites totarol acetate (13) and 6-dehydrototarol (14) were obtained. The metabolite 14 was found to be new as deduced on the basis of spectroscopic techniques. This compound (12) has not been used before for the biotransformation using the plant cultures. It was observed that when artesunate (15) was incubated with O. sanctum cell suspension culture for 10 days, it produced metabolite artemisinin (16). When Sclareol (17, diterpene) was incubated with rapidly growing cell suspension cultures of O. sanctum, it afforded two known compounds 3-keto sclareol (18) and 3β, 3-hyrdroxy sclareol (19), but this is a new route for these biotransformed products. When humulene (sesquiterpene) was incubated with rapidly growing cell suspension cultures of O. basilicum, it afforded a known compound 2,6-diepoxy humulene (22), but this is a new route for this biotransformed product. Bioassay studies were also performed for transformed products obtained through biotransformation. The activities employed were anticancer, antioxidant and enzyme inhibition studies. Transformed compounds did not exhibit any remarkable activity as compared to substrates. To evaluate the diversity of plant cell cultures for biotranformation studies, protoplast isolation and fusion method was tested. For protoplast isolation, in vitro developed leaves and callus of O. sanctum and O. basilicum were used as source tissue. The crucial factors were yield (per ml) and viability of protoplasts for successful protoplast isolation. So, at the optimized enzyme concentration of 0.5 % each of Cellulase R-10 and Macerozyme R-10, the 12 hour incubation is sufficient for optimum protoplast yield (5 x 105 cells/ml) and viability (85%). These isolated protoplasts were then cultured on BH3 + KL medium containing 2,4-D (0.5 mg/L) and NAA (2 mg/L). The further subculturing initially on solid MS medium supplemented with 2,4-D with Kinetin for four weeks, and then BAP 0.1 mg/L and IAA 0.05 mg/L for four more weeks resulted in plantlet formation in O. sanctum only. O. basilicum protoplast did not regenerate into plantlet. The fusion of Ocimum spp. was also performed using PEG and electrofusion method. In electrofusion, the protoplasts of Ocimums did fuse to become a putative hybrid, but they did not survive in culture medium after two weeks of culture. As for PEG fusion method, protoplast did not successfully hybridize and eventually died. The hairy root culture was then established to evaluate their potential for biotransformation and comparison studies were conducted. Hairy root culture was developed by co-cultivating Agastache anisata leaf and stem with root inducing bacterium, Agrobacterium rhizogenes. The induced hairy roots and normal roots were cultured in liquid medium and compound (17) was incubated that yielded biotransformed product (19). However, hairy root culture produce high yield (almost twice) and utilized the substrate (17) completely in 20 days as compared to normal root suspension culture. Finally, tomato (Lycopersicon esculantum) transgenic (expressing Hepatitis B surface antigen) and non-transgenic callus, and suspension culture were employed for biotransformation for comparison studies. Both cultures transformed the substrate (17) yielding metabolite 19. There was no significant difference in biotransformation ability.
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110. An-Nasr/Help

110. An-Nasr/Help

I/We begin by the Blessed Name of Allah

The Immensely Merciful to all, The Infinitely Compassionate to everyone.

110:01
a. When Allah’s help arrives and HE opens up your way to victory after victory,

110:02
a. then you see people entering Allah’s Religion of Islam en-mass, in swarming crowds.

110:03
a. So glorify your Rabb - The Lord with HIS Praise,
b. and seek HIS Forgiveness.
c. Surely HE is the Acceptor of Repentance and Ever-Pardoning.

احادیث ِنزو لِ مسیح ابن مریم علیہما السلام میں قادیانیوں کی تاویلات کا تنقیدی جائزہ A Critical Review of Interpretations of Qādiyānies in Aḥadīth of Nuzūl Al-Masīḥ Ibn e Maryam (علیہما السلام)

The negation of the crucifixion and murder of Maseeh علیہ السلام and the ascension to heaven are mentioned in the Holy Qur'an and the revelation to the earth is mentioned in the authentic Ahadith. The Holy Qur'an and Hadith do not contradict each other on the ascension and revelation of Maseeh علیہ السلام. Mirza Ghulam Ahmed Qadiyani interpret those thirty verses of the Holy Qur'an are about the death of Maseeh علیہ السلام which have nothing to do with his death. The Hadiths of the revelation of Maseehعلیہ السلام benefit from the Holy Qur'an, not derived from any Ijtihad or Israelism. The word death is not mentioned for Essa علیہ السلام in the Holy Qur'an. It is not possible for the Hadith to describe the return of Essa علیہ السلام to earth before the Day of Judgment and let his death be mentioned in the Holy Qur'an. Otherwise, instead of stating the meaning of Holy Qur'an, the Hadith will contradict the Qur'an. The Qur'an mentions the ascension of Essa علیہ السلام to heaven and the Hadith describes the revelation to the earth. There is not a single Hadith that indicates that Essa علیہ السلام refers to Mirza Ghulam Ahmed of Qadian and his descent means that is born from the mothes’s womb, or it is a descent as buroz (بروزی).

Analysis of Alternative Extension Approaches to Technology Dissemination and its Utilization for Sustainable Agricultural Development in the Punjab, Pakistan

Agriculture is the single largest sector and dominating driving force for growth and development of Pakistan‟s economy. Its share in GDP is 24% and employs 48.4% of the total work force of the country. It also provides raw materials for the agro-based industries and adds sustainability to Pakistan‟s export earnings. At present the average yields of various crops are low as compared to their potential yields and also to those of the advanced countries. The research advancements made in the field of agriculture indicate that the available technologies have much potential, which is not being properly exploited by the farmers. The research studies indicate that lack of proper dissemination of agricultural technologies among the farmers is still of one of the major causes for their non-adoption by the farmers. For the effective dissemination of agricultural technologies among the farmers, many extension approaches have been implemented from time to time. At present three main approaches are working in the country: public sector extension approach (PSEA), participatory extension approach (PEA) and commodity specialized extension approach (CSEA) for the guidance and education of the farmers, but still the extension‟s role does not appear to be much effective. However, each approach has some strengths, which may be used by other approaches for the improvement of extension work. Keeping in view this situation, the present study was planned to analyze these three approaches in the context of technology dissemination and its utilization by the farmers. For this purpose the data were collected from 360 respondents taken from the farmers of the three selected approaches by selecting an equal sample of 120 respondents each, from 10 randomly selected villages of tehsil Chak Jhumra of Faisalabad district in which the three approaches are simultaneously in operation. A comparison of the three selected approaches was made against the selected parameters like sustainability, farmers‟ participation, women participation, linkages development, knowledge gain, technology utilization, diversification, competence of extension field staff (EFS), effectiveness of extension messages, effective use of extension methods, responsiveness to various categories of rural people, organization of target groups, responsiveness to national policies and goals and overall perception of the farmers regarding these approaches. The collected data were analysed by using frequency distributions, weighted scores, analysis of variance, standard variation and LSD values. The Chi-square was also used for computing relationship between the socio-economic characteristics of therespondents and some dependent variables like farmers‟ participation, knowledge gain and technology utilization. The results indicated that PSEA was a better approach than PEA and CSEA with regard to the introduction of sustainable agricultural practices among the farmers. PEA had provided more opportunities to the farmers for their participation in extension programme development than the PSEA and CSEA. PSEA and CSEA had absolutely provided no opportunity for women participation at any stage of extension programme development whereas the women were given opportunities for their participation in PEA. PSEA had developed more linkages with various categories of farmers and agricultural allied agencies than those of PEA and CSEA. PEA was the approach that had covered all the diversified areas whereas rest of the two approaches were not putting sufficient efforts in providing coverage. PEA was a better approach than the other two approaches (PSEA and CSEA). PSEA was ranked at the top with regard to the knowledge gain regarding crop production and protection practices by the farmers, whereas PEA and CSEA were rated as 2 nd and 3 rd respectively. As regards the technology utilization by the farmers PSEA, PEA and CSEA were rated 1 st , 2 nd and 3 rd respectively. PSEA got the highest score among the selected approaches with regard to competence of extension field staff. The extension messages were well prepared in PSEA than those of PEA and CSEA. PEA was responsive to more categories of rural people as compared to the other two approaches (PSEA and CSEA). The EFS of PSEA used the extension methods more effectively than those of PEA and CSEA. The EFS of PEA was more serious in organizing the target groups among the rural communities than the other two approaches (PSEA and CSEA). According to the overall perceptions of the farmers, the PEA was a better approach followed by the PSEA and CSEA.