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Rna Interference Based Resistance Against Chilli Leaf Curl Disease Complex

Thesis Info

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Author

Shafiq, Muhammad

Program

PhD

Institute

Quaid-I-Azam University

City

Islamabad

Province

Islamabad.

Country

Pakistan

Thesis Completing Year

2014

Thesis Completion Status

Completed

Subject

Biotechnology

Language

English

Link

http://prr.hec.gov.pk/jspui/bitstream/123456789/10209/1/Muhammad%20Shafiq_Biotech_2014_QAU_PRR.pdf

Added

2021-02-17 19:49:13

Modified

2024-03-24 20:25:49

ARI ID

1676727106112

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Chilli (Capsicum annuum), a member of the family Solanaceae, is an important spice crop cultivated in tropical and subtropical countries. Chilli leaf curl disease (ChLCD) is a limiting factor for chilli yield across Pakistan and India. Symptoms of ChLCD include severe upward leaf curl with cup-shape, yellowing and stunted plant growth. This disease is caused by begomoviruses (single-stranded DNA viruses (family Geminiviridae) that are transmitted by whiteflies). All three different types of begomoviruses are already reported from chillies. In this study chilli samples showing typical disease symptoms were collected from Faisalabad in the Province of Punjab (Pakistan) during the year 2006. All samples were positive for begomoviruses and Pepper leaf curl Lahore virus (PepLCLV) along with Tomato leaf curl New Delhi virus DNA B and Chilli leaf curl betasatellite (ChLCB) were identified. The DNA of Pepper leaf curl Lahore virus consisted of 2747 nucleotides and had the highest sequence identity (99%) with PepLCLV-[PK: Lah: 04] AM404179). Agrobacterium-mediated inoculation of the partial repeat construct of PepLCLV clone obtained in this study to Nicotiana benthamiana induced very mild symptoms and very low flow of viral DNA were detected in infected plant leaves. Co-inoculation of ChLCB with PepLCLV to N. benthamiana did not affect the symptoms severity or the virus titre. However neither the PepLCLV alone or with ChLCB was able to induce any symptoms on N. tabacum L. and C. annuum. Inoculation of PepLCLV with DNA B of ToLCNDV induced very severe symptoms in N. benthamiana, N. tabacum and symptoms typical of ChLCD in C. annuum. Southern hybridization analysis showed very high DNA accumulation for PepLCLV and DNA B of ToLCNDV in all three plant species. Sequence analysis showed that predicted rep-binding iterons in PepLCLV (GGGGAC) was different with two nucleotides from that of ToLCNDV DNA B (GGTGTC). This indicated tolerance of two nucleotide differences in iterated elements for replication. Based on this study, it is proposed that PepLCLV has been recently mobilized into chillies upon its interaction with DNA B of ToLCNDV. This is the first experimental demonstration of infectivity for a bipartite begomovirus causing ChLCD in chillies from Pakistan and suggests that component capture may contribute to the emerging complexity of begomovirus diseases in the region. NIBGE |School of Biotechnology NIBGE Faisalabad [QAU Islamabad] iv The purpose of this study was to develop a broad-spectrum resistance against ChLCD complex based on the concept of pathogen-derived resistance. A hairpin RNAi construct (peAC1-AC2dsRNA/pFGC) based on overlapping region of highly conserved region of Rep and TrAP of PepLCLV was produced in a binary vector pFGC5941. In order to study silencing efficiency of peAC1-AC2dsRNA/pFGC, the construct was transiently challenged with PepLCLV along with DNA B ToLCNDV. Results showed that the RNAi construct was successful in blocking the viral infection as all tested plants were symptomless. Transgenic tobacco plants expressing this construct challenged with PepLCLV and DNA B of ToLCNDV by agroinoculation and with viruliferous whiteflies showed variable resistance ranging from 6.6% to 93.3%. Lines showing resistance more than 75% were ranked resistant/tolerant while lines showing resistance less than 50% were ranked susceptible. One line TA14 showing 93.3% was ranked as highly resistant/tolerant while the line TA 3.2 showing 6.6 % resistance/tolerance was ranked as highly suscepteible. These lines also exhibited significant resistance against ToLCNDV. The relatively conserved nature of Rep and TrAP and their ability to help in development of resistance against heterologous virus suggested that the technology may be useful to develop broad-spectrum resistance. Plants need broad spectrum resistance because they were often infected with multiple begomoviruses in the field. Some viral proteins interfere with different cell signalling pathways and induce symptoms in plants. For example expression of P6 protein of CaMV in Arabidopsis induced dwarfness in transgenic plants. It is reported that Arabidopsis plants with TIR3 gene mutated (tir3) are also dwarf. P6 transgenic (A7, B6) and tir3 Arabidopsis plants which were resistant to auxin and ethylene also showed resitance to 2,3,5-Triiodobenzoic Acid (TIBA) treatment. It indicates that P6 interacts with a pathway overlapped with TIR pathway. Symptoms in Arabidopsis expressing the P6 protein of CaMV probably comes by disturbance of auxin response factor 10 (ARF10), ARF16, and ARF17 also. Also P6-expressing transgenic Arabidopsis plants showed reduced accumulation of miR160 which is known to regulate ARF10, ARF16 and ARF17. A protocol was also developed for chilli plant regeneration using hypocotyl and cotyledon explants. The study was conducted to observe the effect of genotypes, culture conditions and growth regulators on plant regeneration of chili pepper (C. NIBGE |School of Biotechnology NIBGE Faisalabad [QAU Islamabad] v annuum) genotypes grown in Pakistan including Seedex Pepper (SP), Loungi, Tatapuri and Sanam. Of the evaluated genotypes, SP was found to be the most responsive for both hypocotyl and cotyledon explants. Hypocotyl and cotyledon explants were tested for transformation by A. tumefactions LBA4404 having the 35S GFP/pFGC construct and A. tumefaciens EHA105 with peAC1-AC2dsRNA/pFGC construct. Co-cultivation at different temperatures (22 and 25ºC), photoperiods (16h light 8h dark, and complete darkness) as well as co-cultivation time periods, were evaluated. GFP assays showed that putative transgenic calli had not been transformed and calli died after 40-60 days. The experiment was repeated ten times. The data presented in this thesis should help in devising novel control strategies against Begomoviruses. A combination of novel sources of resistance with natural sources of resistance may help to exploit the technology in the field conditions. However, because most pepper varieties are recalcitrant to genetic transformation, control of diseases caused by the ChLCD complex using this strategy awaits future progress.
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سِک ماہی دی

سِک ماہی دی
جہلم دے دریا دا ٹھنڈا ٹھنڈا پانی اے
لَے اللہ دا ناں جے کشتی پار لنگھانی اے
دلبر ساتھوں دور ہے وسدا
ناز ، ادا تھیں دلڑی کھسدا
رستہ رب رسولؐ دا دسدا
آجا در تے چھیتی جے قسمت ازمانی اے
لَے اللہ دا ناں جے کشتی پار لنگھانی اے
دلبر دی میں دید دی پیاسی
دلبر باہجھوں رہے اداسی
کدوں اوہ سوہنا مکھ وکھاسی
ہک دن ماہی اِن شاء اللہ دید کرانی اے
لَے اللہ دا ناں جے کشتی پار لنگھانی اے
دلبر یار دا شہر سنگوئی
جس دی جگ وچ ریس ناں کوئی
پیر اصغر دی دیو دھروئی
جس نے اُس دا ورد پکایا اوہو مرد گیانی اے
لَے اللہ دا ناں جے کشتی پار لنگھانی اے

جہلم شہر ہے بڑا رنگیلا
اوگنہاراں دا اے وسیلہ
اوتھے جان دا کر کجھ حیلہ
عیباں والڑیاں دی تے ہو بخشش جانی اے
لَے اللہ دا ناں جے کشتی پار لنگھانی اے
ٹلے جوگی ریت بنائی
رانجھا آیا تلک لگائی
چنڑی رنگدیاں دیر نہ لائی
سجناں ریت قلندری ایہا بہت پرانی اے
لَے اللہ دا ناں جے کشتی پار لنگھانی اے
’’ڈھوک رجو‘‘ ہے پنڈ نرالا
جتھے وسدا اللہ والا
میلے دلاں نوں کرے اُجالا
کامل اکمل سوہنا مرشد جس دا فیض روحانی اے
لَے اللہ دا ناں جے کشتی پار لنگھانی اے

سد الذرائع حقيقته ونماذج من تطبيقاته المعاصرة

Today’s world is a global village. Societies affect each other far deeper and faster than ever. New problems are sprouting with every coming day. We feel that the cultural issues are the most significant ones in this context. The question is how we should deal with these problems. To us, the solution lies in the concept of Sadd al-Dharā’i‘(prevention), one of the instruments of Sharī‘ah (Islamic Law). Such is its importance as all the four schools of thought are of the same opinion about its scope. A cultural change if drives Muslims away from the objectives of Sharī‘ah, it will have to be stopped or altered to suit the objectives, but if it leads to something good without damaging the objectives of Sharī‘ah, it will be accepted. New problems are multifarious. We need to bring ijtihād into practice and solve such problems. This research article discusses the concept of Sadd al-Dharā’i‘(prevention.), its meaning, definition, and scope. It also discusses different opinions of scholars. The author of this paper, then, deliberates its use for its applications to solve the new problems being faced by the Muslims across the world.

Foodrive

Each year, an estimated 36 million tonnes of food, which accounts for 40% of all the food produced in Pakistan, is wasted [1]. Yet, the staggering amounts of food waste witnessed at restaurants and wedding halls continues unceasingly. The problem might be spearheaded by a few corporations but it is driven by the consumption patterns of millions of people, particularly those residing in the urban centers. Therefore, we believe any solution to reducing food wastage and ending hunger must come through a collective effort of those very people. With the mass adoption of mobile technology in recent years, such a solution is now very much possible and this project aims to make that a reality. In this project we are developing an android based application that would handle the tasks of contribution, collection, verification, and distribution of food from restaurants, parties and individuals to organizations that distribute food or directly to the hungry