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Home > Expression Analyses, Efficacy Studies and Environmental Risk Assessment of Insecticidal Proteins Produced in Transgenic Plants

Expression Analyses, Efficacy Studies and Environmental Risk Assessment of Insecticidal Proteins Produced in Transgenic Plants

Thesis Info

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Author

Ullah, Inaam

Supervisor

Tariq Mahmood

Program

PhD

Institute

Pakistan Institute of Engineering and Applied Sciences

City

Islamabad

Province

Islamabad

Country

Pakistan

Thesis Completing Year

2015

Thesis Completion Status

Completed

Subject

Natural Sciences

Language

English

Link

http://prr.hec.gov.pk/jspui/bitstream/123456789/6802/1/PhD%20Thesis%20Inaam%20Ullah%2010-7-1-006-2008.pdf

Added

2021-02-17 19:49:13

Modified

2024-03-24 20:25:49

ARI ID

1676726168239

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Insect pests are a serious threat to agricultural crops all over the world as they cause huge losses to crop yield and economy. Due to low innate immunity in plants, synthetic insecticides have been used to cope with the pest problem since decades. These chemicals are expensive, broad spectrum and hazardous to health and environment. Moreover, most of the target insect species have developed resistance against the available insecticides. Therefore, search for alternative methods of insect pest control has been a priority in agricultural research. One of the successful methods is to develop Insect Resistant, Genetically Modified Crops (IR-GMCs) expressing insecticidal proteins. IR-GMCs have been rapidly adopted around the world as they are target specific, environment friendly, cost effective and easy to use. However, two major risks of growing IR-GMCs have been proposed; resistance development against insecticidal toxins (e.g. Cry proteins) expressed in plants and adverse effects to beneficial, nontarget arthropods (NTAs). NTAs include important species that contribute directly to agricultural ecology via biological control, pollination and decomposition of organic materials. Pakistan officially adopted Bt-cotton in 2010 and became one of the leading countries in terms of area under cultivation by 2013. Variation in the expression of Bt proteins have been associated with reduced efficacy of the IR crops and also resistance development in the target insect pests. Therefore, one of the objectives of this study was expression profiling of first batch of approved, locally bred Bt-cotton varieties through ELISA and efficacy analysis against Helicoverpa armigera. While Bt cotton varieties are effective in controlling several key lepidopteran pests, they are less effective against some Lepidoptera species and provide no protection against non-lepidopteran pests. In addition, the initial cases of resistance to Cry1Ac have been observed in target insect pests in many countries. It is thus indicated to explore alternative source for toxins that can be incorporated in future transgenic plants. The peptide ω-Hexatoxin-Hv1a (Hvt) is one of the most studied spider venom toxins. Its insecticidal potential has been reported for species belonging to different arthropod orders including Lepidoptera, Diptera and Orthoptera. The gene encoding for Hvt has thus been transformed into cotton and tobacco to protect the plants from damage by Lepidopteran pests. The second objective of the study therefore, was to evaluate the expression of ω-Hexatoxin-Hv1a gene in transgenic plants, and the toxicity of plantAbstract xi expressed and purified Hvt against target lepidopteran insects and four non-target arthropod species. Bollgard IITM cotton plants and purified Cry2Ab2 protein were included in the study as comparators. In studies with locally bred Bt cotton, expression of Cry1Ab/1Ac protein were found to vary significantly (P<0.05) among varieties and across different sampling dates. The highest mean expression was recorded in GN-31 and Sitara-008 and the lowest in FH- 113 and MG-6 while across sampling dates the highest mean expression was recorded at 30 days after emergence (DAE) which decreased along the season with lowest mean at 120 DAE. The control of the H. armigera was directly proportional to the amount of protein expressed in the leaves. Critical level of Cry1Ab/1Ac in leaves was found to be 770±25 ng g-1 for 95% control of the target insect pests. In studies with Hvt plants, 100% larval mortality was observed on Hvt-transgenic tobacco line T-21 and cotton line T-7 plants but not on cotton line T-10. The likely reason was the significantly lower expression level in the transgenic cotton line T-10. When fed on artificial diet containing purified Hvt protein, LC95 values for Spodoptera littoralis and Heliothis virescens were found to be 28.31 and 27.57 μg mL-1, respectively. Non-target studies were performed with two predatory species, larvae of Chrysoperla carnea and Coccinella septempunctata, adult females of the aphid parasitoid Aphidius colemani, and adult workers of honey bees Apis mellifera. Orally administered Hvt protein even at a concentration of 40 μg mL-1 did not affect a number of life-table parameters of tested NTAs. Similarly purified Cry2Ab2 did not cause adverse effects on any of the non-target species tested at a concentration of 10 μg mL-1. However, detailed molecular work is required to determine the fate of orally administered Hvt protein in these species. Hence, Hvt might be an interesting candidate for developing insecticidal plant varieties to control pest Lepidoptera. However, the concentration at which the protein is available in the plants appears to be critical in determining its efficacy against target pests. Therefore, breeders and biotechnologists must combine genotypes and promoters promising higher expression of insecticidal proteins. Furthermore, detailed risk assessment studies may be conducted before commercial release of Hvt cotton and tobacco.
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109. Al-Kafirun/Those who knowingly deny

109. Al-Kafirun/Those who knowingly deny

I/We begin by the Blessed Name of Allah

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

109:01
a. Say:
b. ‘O you who knowingly deny the truth of Islam!

109:02
a. I will not submit in awe and worship to what you worship,

109:03
a. nor will you worship the One WHOM I submit in awe and worship – Allah, The One and Only God.

109:04
a. And neither have I ever submitted in awe and worship to what you worship,

109:05
a. nor have you ever worshiped the One WHOM I submit in awe and worship,

109:06
a. Therefore, for you, is your religion and its consequential ramifications in the Sight of Allah,
and
b. for me, is my religion’ and its consequential ramifications in the Sight of Allah.

علم اسباب ورود الحدیث: ایک تحقیقی جائزہ

Asbab-e-worood-e-Hadith means the context of background of a Haith. It is impossible to understand the original myth of hadith without knowing its background and context. This Article presents the concept of asbab-e-worood-e-Hadith. The discussion has been premeditated to explore the meanings and importance of asbab-e-worood. In this regard, keeping in view the nature of topic, the guidance has been sought from the Ahadith of Holy Prophet (PBUH). Asbab-e-worood in its nature having similarities with asbab-e-nazool-e-Quran. This article also describes the relationship between asbab-e-worood-e-hadith and asbab-e-nozool-e-Quran. In this regard some examples have been discussed also. This article also describes how asbab-e-worood is important to specify the meanings of text and explain the textual ambiguities. Key Words: Asbab-e-worood-e-Hadith Asbab-e-Nozool-e-Quran Sabab-e-worood qasasia 4.         Sabab-e-worood sawalia

Molecular Genetic Studies for Drought Tolerance in Cotton

Although water-limited environment is detrimental to cotton growth and productivity worldwide, development of drought tolerant cotton genotypes may improve yield in drought prone areas. The present study was aimed to examine drought tolerance of a set of Upland cotton genotypes using both empirical as well as analytical approaches, and molecular mapping of the traits conferring drought tolerance. Two field experiments and one greenhouse study were conducted in 2003 and 2004, and performance of 32 cotton genotypes for different physiological attributes conferring drought tolerance, and productivity traits were recorded under well-watered (W 1 ) and water-limited (W 2 ) regimes. Seedcotton yield (SCY) and its components were markedly affected under W 2 regime. Mean reduction in SCY due to water deficit was 20 and 43% in 2003 and 2004, respectively. Genotypes differed considerably for relative SCY losses due to water stress ranging from 20 to 74%. SCY sustainability under W 2 regime was mainly attributed to maintenance of higher boll number (BN) rather than boll weight (BW). Substantial genotypic variation for gas exchange attributes {(photosynthetic rate (P n ), stomatal conductance (g s ), and transpiration rate (E)}, osmotic adjustment (OA) cell membrane stability (CMS) existed among cotton genotypes. Water stress caused a significant reduction in gas exchange parameters in 2003 and 2004. The positive association (P<0.01) between P n and g s in both years in W 2 regime suggests a major role of stomatal effects in regulating leaf photosynthesis under water-limited conditions. P n and OA were significantly correlated with SCY (P<0.01) in W 2 regime, however, the level of associations of CMS with productivity traits was not significant. Results of green house experiment conducted to ascertain root traits in six selected genotypes demonstrated that drought tolerant genotypes possessed long tap root compared to susceptible genotypes. These findings tend to support the hypothesis that higher photosynthetic rate, maintained through OA and deep root system, leads to sustain SCY under water deficit environment. Therefore, P n and OA may be useful as selection criteria in breeding programs with the objective of improving drought tolerance and SCY under water-limited environments. For genetic analysis of drought tolerance, F 2 and F 2:3 mapping populations derived from a cross of Upland cotton genotypes RH-510 (drought tolerant) and FH-901 (drought susceptible) were evaluated for four physiological attributes, and six productivity traits, - xiv -Abstract respectively. Parent genotypes were selected on the basis of their diverse performance in screening experiments. Significant variation was found for all the traits measured except BW. Correlation analysis revealed significant association (P<0.01) of P n with g s and OA under water stress. A strong relationship (P<0.01) of SCY was found with BN in both the water regimes. Continuous variation pattern of F 2 plants and F 2:3 families for all the traits indicated that measured traits were quantitatively inherited. Transgressive segregation observed in both directions indicated that both the parents transmitted favourable alleles for each trait. Eight hundred and twenty two SSR primer pairs and 520 RAPD primers were surveyed on the genotypes which yielded 65 polymorphic loci including 33 SSRs, 30 RAPDs and two CAPSs. RAPD analysis exhibited comparatively high polymorphism (5.8%) compared to that of SSRs (4.7%). All the 65 markers were assayed on 143 F 2 plants; however, data of 51 loci were utilized for map construction due to ease in allele scoring. Linkage analysis resulted in mapping of 45 loci (24 SSRs, 20 RAPDs, one CAPS) on 10 different linkage groups. The remaining 6 markers were unlinked. Six of the linkage groups were assigned to five chromosomes of the tetraploid cotton genome. The genetic map spanned a total of 697.9 cM, covering around 15% of the total cotton genome with average inter-locus distance of 15.5 cM. QTL analysis mapped 26 QTLs impacting nine physio-economic traits. Genetic analysis of physiological traits under water-deficit stress using interval mapping (IM) and composite interval mapping (CIM) methods collectively detected nine putative QTLs, ranged from one to four for each trait. The QTL QP n 5cC located on chromosome 5 accounted for the largest phenotypic variance of 28% for P n . Interval mapping employed to determine chromosomal location of genes impacting the productivity traits yielded 12 QTLs for five traits in both water regimes. Five additional QTLs controlling these traits were identified using CIM. The information regarding QTLs discovered for the traits conferring drought tolerance, especially those explaining large amount of variation for net photosynthetic rate and osmotic adjustment, may complement breeding efforts to breed for drought tolerance in Upland cotton. Since this study constitutes first knowledge of identification of QTLs for drought tolerance in Upland cotton using F 2 and F 2:3 mapping populations, the identified QTLs need to be validated across different populations and environments before their use in marker assisted selection.