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In Vitro Re Generation and Marker Assisted Evaluation of Genetic Fidelity in Endangered Tree Species Moringa Peregrina (Forsk) Fiori Salama Mohammed Al Dhaberi

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In Vitro Re Generation and Marker Assisted Evaluation of Genetic Fidelity in Endangered Tree Species Moringa Peregrina (Forsk) Fiori Salama Mohammed Al Dhaberi United Arab Emirates University Scholarworks@UAEU Theses Electronic Theses and Dissertations 5-2016 In Vitro Re Generation and Marker Assisted Evaluation of Genetic Fidelity In Endangered Tree Species Moringa Peregrina (Forsk) Fiori Salama Mohammed Al Dhaberi Follow this and additional works at: https://scholarworks.uaeu.ac.ae/all_theses Part of the Agriculture Commons Recommended Citation Al Dhaberi, Salama Mohammed, "In Vitro Re Generation and Marker Assisted Evaluation of Genetic Fidelity In Endangered Tree Species Moringa Peregrina (Forsk) Fiori" (2016). Theses. 453. https://scholarworks.uaeu.ac.ae/all_theses/453 This Thesis is brought to you for free and open access by the Electronic Theses and Dissertations at Scholarworks@UAEU. It has been accepted for inclusion in Theses by an authorized administrator of Scholarworks@UAEU. For more information, please contact [email protected]. • tf'�)) a.ul.o..lI � J.Sl-11 L.JIJ La� I ii5t.o f.? wu:u '\:;/ United Arab Emirates University United Arab Emirates University College of Food and Agriculture Department of Aridland Agriculture IN VITRO REGENERATION AND MARKER ASSISSTED EVALUATION OF GENETIC FIEDILITY IN ENDANGERED TREE SPECIES MORINGA PEREGRINA (FORSK) FIORI Salama Mohanm1ed Al Dhaberi This thesis is submitted in partial fulfillment of the requirements for the degree of Master of Science in Horticulture Under the Supervision of Dr. Shyam S. Kurup May 2016 ii Declaration of Original Work I, Salama Mohammed AI Mutawa AI Dhaheri, the undersigned, a graduate student at the United Arab Emirates University (UAEU), and the author of this thesis entitled "In vitro Regeneration and Marker Assisted Evaluation of Genetic Fidelity in Endangered Tree Species Moringa Peregrina (Forsk) Fiori", hereby, solenmly declare that this thesis is my own original research work that has been done and prepared by me under the supervision of Dr. Shyam Kurp in the College of Food and Agriculture at UAEU. This work has not previously been presented or published, or formed the basis for the award of any academic degree, diploma or a similar title at this or any other university. Any materials borrowed from other sources (whether published or unpublished) and relied upon or included in my thesis have been properly cited and acknowledged in accordance with appropriate academic conventions. I further declare that there is no potential conflict of interest with respect to the research, data collection, authorship, presentation and/or publication of this thesis. Date: /21/6/QO!b iii Copyright © 2016 Salama Mohammed AI Dhaheri All Rights Reserved iv Advisory Committee 1) Advisor: Dr. Shyam S. Kurup Title: Associate Professor Department of Aridland Agriculture College of Food and Agriculture 2) Co-advisor: Dr. Abdul Jaleel Cheruth Title: Associate Professor Department of Aridland Agriculture College of Food and Agriculture v Approval of the Master Thesis This Master Thesis is approved by the following Examining Committee Members: 1) Advisor (Committee Chair): Dr. Shyam S. Kurup Title: Associate Professor Department of Aridland Agriculture College of Food and Agriculture . �----! --.r--P Stgnature -�� �----- ---- Date --------- 2) Member: Dr. Abdul Jaleel Cheruth Title: Assistant Professor Department of Aridland Agriculture College of Food and Agriculture Signature Date 3) Member (Ext;J: Examiner): Dr. Amad Mohammed Jamil Al- Azzawi Title: Chairperson of Pharmaceutical Chemistry RAK Medical & Health Science University, Ras al Khaimah, UAE Signature Date c:Llt ( ) /-"Vj G vi This Master Thesis is accepted by: Dean of the College of Food and Agriculture: Professor Afaf Kamal-Eldin a Signature __f/)-f -fh(j9 +-�-+-d), L.!,",/I V-c..=CZJ,G, :...:::(.._..J�.t·lJ-..-__-== =- =-- Date Dean of the College of the Graduate Studies: Professor Nagi T. Wakim t:lf: A� Signan'' · I ·- Copy lf2_of __l_D vii Abstract Moringa peregrina is a native plant in the United Arab Emirates; it is called “Shua”. It is an endangered species with important nutritive and medicinal properties. It shows high adaptability to the arid condition with short germination time after irrigation. Unmanaged grazing and slow regeneration rate after browsing leads to the endangerment of the species causing erosion in biodiversity. One way to protect these rare species is by multiplying the planting material through tissue culture system and re-introducing at the habitat. Microproagation is an in vitro method used for plant clonal multiplication under controlled condition. This technique allows conserving the genetic characteristics of the plants which are usually affected by environmental restrictions. In the present study different parts of M. peregrina explants will be used for standardizing the mass propagation. Direct organogenesis of nodal explants collected from the in vitro germinated seedlings were inoculated in MS media supplemented with different hormone treatment (TDZ, BA and NAA) with several concentrations. Thidiazuron treatment in the media observed to be better than BA and NAA combinations. It has been observed that media supplemented with TDZ 1 µM induced an average of 2.4 axillary shoots per explant. For callusing the results show that MS media supplemented with 2,4-D 2 μM/l and TDZ 0.5 μM/l found to have the highest callusing percentage and size. The rooting of in vitro developed plantlets obtained from nodal ex plants were induced through media supplemented with BA in combination with NAA and IBA. The number of roots was high in BA and NAA medium compare to BA and IBA. The genetic fidelity of the clones produced from viii the in vitro protocol was assessed using molecular markers. The similarity index between the progenies and parent was above 94.9%.This shows that in vitro developed plantlets are identical with its mother plant. This will facilitate standardizing future conservation programs to limit the erosion of the genetic biodiversity in Moringa peregrina and similar species. Keywords: In vitro propagation, Moringa pergrina, endangered tree species, ISSR analysis. ix Title and Abstract (in Arabic) اﻹكثار النسيجي المخبري للنبات المهدد باﻹنقراض )مورينغا بيريجرينا( وتقييم المادة الوراثية للنباتات التي تم إنتاجها في المختبر بإستخدام بروتوكول الواسمات الجزيئية الملخص (Moringa peregrina ) المورينغا بيريجرينا هو من أهم النبات اﻷصلية المحلية في دولة اﻹمارات العربية المتحدة؛ يطلق عليه إسم "الشوع". و هو من اﻷنواع المهددة باﻻنقراض و هو المعروف بخصائصه الغذائية والطبية الهامة. و للمورينغا بيريجرينا القدرة العالية على التكيف والنمو في الظروف القاحلة حيث ان لها القدرة على اﻹنبات بعد مدة قصيرة من الري. و يعتبر الرعي الغير منظم ومعدل التجديد البطيئ هو من أهم اﻻسباب التي تؤدي إلى تعريضها للخطر و تسبب تآكل في التنوع البيولوجي. هناك عدة طرق لحماية هذه اﻷنواع النادرة من اﻹنقراض من ضمنها اكثارها من خﻻل نظام زراعة اﻷنسجة ثم إعادة زراعتها في بيئتها الطبيعية. (Microproagation) هو طريقة في المختبر تستخدم ﻷكثار النسيج النباتي تحت ظروف خاضعة للرقابة. هذه التقنية تتيح الحفاظ على الخصائص الجينية للنباتات التي تتأثر عادة بسبب القيود البيئية. في هذه الدراسة سيتم إستخدام أجزاء مختلفة من اﻻنسجة العقدية للنبات و التي سيتم زراعتها في البيئة الغذائية MS و ستم معالجتها بتركيزات وتركيبات مختلفة لبعض الهرمونات مثل (TDZ, BA, NAA) بغرض اكتشاف البيئة الغذائية اﻷكثر مﻻئمة لعملية اﻷكثار النسيجي المخبري.وقد لوحظ أن البيئة الغذائية المزودة بمادة TDZ و بنسبة 1 ميكرومول نجحت في انتاج براعم إبطيه بمتوسط 4.2 للنسيج الواحد المزروع في البيئة الغذائية .أظهرت النتائج أيضا أن البيئة الغذائية المناسبة ﻹنتاج )callus) هي )MS ( مزودة ب 4 ميكرومول / لتر من هرمون D-2,4 و0.5 ميكرومول / لترمن هرمون TDZو التي أدت الى أعلى نسبة) callusing( و أكبرها حجما. البيئة الغذائية المثالية التي أستخدمت ﻹنتاج الجذور تحتوي على (BA و NAA وIBA ) . وقد لوحظ زيادة عدد الجذور في البيئة التي تحتوى على (BA) و (NAA) مقارنة بتلك التي تحتوي على (BA) و)IBA). تم تقييم المادة الوراثية للنباتات التي تم إنتاجها في المختبر باستخدام بروتوكول الواسمات الجزيئيةوتبين أن مؤشر التشابه بين السﻻﻻت واﻷم يفوق 92.9٪ مما يظهر تطابق السﻻﻻت x التي تم إنتاجها في المختبر مع النباتات اﻷم . و من الجدير بالذكر أن هذه الدراسة ستساعد في تسهيل و توحيد برامج المحافظة المستقبلية للحد من تآكل التنوع البيولوجي الوراثي في المورينغا بيريجرينا واﻷنواع المماثلة. مفاهيم البحث الرئيسية: اﻷنسجة النباتية، مورينقا بيريجرينا، النباتات المهدده باﻹنقراض، بروتوكول الواسمات الجزئية. xi Acknowledgements First of all, I would like to thank Allah, without his grace and care I wouldn’t be where I am today. I would like to express my deep appreciation for the leaders of my country, ‘the United Arab Emirates’, who provided all I needed to complete my studies. I would like to thank my loving family (my Mom Fatima, my husband Mohammed, my brothers (Hamad, Ali, Sultan and Nasser), my sisters (Aisha, Amna, Maryam, Mai and Fatima) and my kids Ahmed and Maryam for always being there, for encouraging me to successes, and for their love and support. My thanks go to all my teachers throughout my life who guided me in my classes and in my life. Many thanks to college of food and agriculture, food science department (Dr. Aisha boushlibi, Dr. Oya, Dr. Adam, Dr. Louis, Dr. Hanan, Mr. Jobe, Mr. Abdulgader and all others) and to the college of chemistry specially Dr. Salman Ashraf. I would like to thank my committee for their guidance, support, and assistance throughout my preparation of this thesis. Special thanks to Mr. Fayas our research assistant. xii Dedication I dedicate my thesis to my late father, my wise mom, my lovely husband and kids. xiii Table of Contents Title ....................................................................................................................................
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