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Improvement of Lentil (Lens Culinaris Medik.) Through Genetic Transformation

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Improvement of Lentil (Lens Culinaris Medik.) Through Genetic Transformation Improvement of Lentil ( Lens culinaris Medik.) through Genetic Transformation Von der Naturwissenschaftlichen Fakultät Der Gottfried Wilhelm Leibniz Universität Hannover Zur Erlangung des Grades einer DOKTORIN DER NATURWISSENSCHAFTEN Dr. rer. nat. genehmigte Dissertation Von M.Sc. Rehana Hashem Geboren am 23.10.1971 in Dhaka, Bangladesh 2007 Referent: Prof. Dr. Hans - Jörg Jacobsen Korreferent: Prof. Dr. Edgar Maiß Prüfungsvorsitz: Prof. Dr. Bernhard Huchzemeyer Tag der Promotion: 23 February 2007 Dedicated to my beloved parents And My respected teachers ABSTRACT Work title: Improvement of Lentil ( Lens culinaris Medik.) through genetic transformation. Hashem, Rehana The future agriculture will depend more on legume crops because they all have high energy and high protein production for human and animal nutrition as well as amino acid profiles complementary to those of other crops, mainly cereals. The unique symbiotic ability of legumes is to use atmospheric nitrogen for plant growth makes them preferable crops for sustainable agriculture. Lentil is the 2nd most important grain legume that gained worldwide economic importance as a source of protein (25.5 – 28.31 %). In addition, it is also suitable as a rotation crop to replenish soil nitrogen levels. It is a crop of cooler temperature and is widely grown in the temperate zones of the world. The production of lentil is usually considerably below the established yield potential as this crop is very sensitive to particular biotic and abiotic stresses. The most serious biotic attribute constrain in lentils are the foliar diseases such as Ascochyta blight, rust, Stemphylium blight and Botrytis grey mold. Yield stability and productivity and the value of lentil could be greatly increased by the introduction of stably inherited traits such as pest and disease resistance, herbicide resistance or improved protein quality. These traits are not available in natural populations of near relatives of cultivated lentils, but current advances in plant genetic engineering provide a potentially powerful tool for achieving these goals by an alternative mean. The aim of the present study is therefore, the establishment of a reproducible and efficient transformation system for Lens culinaris Medik which is suitable for the insertion of agronomically desirable genes to overcome the limitations imposed by traditional breeding process. Along with this, another objective is to explore a simple marker free transformation system. Antibiotic resistance genes (e.g. npt II , hpt ) or herbicide resistance genes (e.g. bar ) are essential for selectively propagating transformed cells and tissues. However, subsequent maintenance of markers is unnecessary. Elimination of markers is advocated since it theoretically can not be excluded that antibiotic or herbicide resistance genes may be transferred to pathogenic bacteria or weeds, although the likelihoods are extremely low. The Bari Musur variety, BM4 was selected through its regeneration performance. Decapitated embryos with single cotyledon discs were selected as explant for transformation. The Agrobacterium strain EHA- 105 with the plasmid pSCP1 was used for transformation. The plasmid was harbouring the selectable marker gene bar , which encodes the enzyme phosphinothricin acetyltransferase (PAT) and a pgip gene from raspberry ( Rubus idaeus L.), I coding for polygalacturonase inhibitory protein. The expression of this recombinant gene can confer resistance against fungal pathogens ( Colletotrichum, Botrytis etc). The total procedure from seed to seed was between 2.5 - 4 months until getting transgenic lentil seeds. Transformation efficiency was found to be about 29%. For assessing the possibilities to develop a marker free transformation system, the bar-gene was removed and PGIP gene was kept in the T-DNA cassette before carrying out transformation work. Transformation with the new construct gave us a transformation success rate of 35% as estimated from the T 0 clones. The first analysis of a transformation rate of 35% will be confirmed by further analysis of the progenies. On the functional level, the plants were analyzed via a semi-quantitative polygalacturonase- inhibition assay. Activity of the pgip gene against Colletotrichum lupini, C. acutatum , Botrytis cinerea was tested. It was shown , the established method could provide a powerful tool to achieve markerfree transgenic lentil plants. Keywords : Agrobacterium , transgenic lentil, polygalacturonase inhibiting protein, antifungal resistance, marker-free transformation. II Zusamenfassung Arbeitstitel : Verbesserung der Linse ( Lens culinaris Medik.) durch genetische Transformation Hashem, Rehana Die Bedeutung von Leguminosen in der Landwirtschaft wird in Zukunft aufgrund ihres hohen Energie- und Proteingehalts für den Bereich der menschlichen und tierischen Ernährung steigen. Zusätzlich enthalten Hülsenfrüchte, im Vergleich zu anderen Feldfrüchten wie z.B. Getreide, ergänzende essentielle Aminosäuren. Die einzigartige Fähigkeit von Leguminosen über die Symbiose mit Rhizobien atmosphärischen Stickstoff für ihr Wachstum zu nutzen, macht sie zu bevorzugten Saaten in der nachhaltigen Landwirtschaft. Durch den hohen Proteingehalt (25,5-28,31%) gehört die Linse zu den wichtigsten Hülsenfrüchten weltweit. Des weiteren spielt sie eine wichtige Rolle in der Fruchtfolge zur Regeneration des Stickstoffs im Erdboden. Angebaut wird die Linse hauptsächlich in gemäßigten Zonen. Aufgrund der hohen Sensitivität gegenüber biotischen und abiotischen Stressfaktoren liegt die Produktivität der Linse oft weit unterhalb des möglichen Ertragspotentials. Eines der größten Probleme beim Linsenanbau stellen Blattfleckenkrankheiten wie Aschochyta und Mycospherella dar, aber auch Rost ( Uromyces ) und Grauschimmel ( Botrytis ), und bodenbürtige Erreger wie Aphanomyces euteiches sind von Bedeutung. Durch die Integration stabil vererbter Merkmale wie Schädlings- und Krankheitsresistenzen, Herbizidresistenz oder erhöhte Proteinqualität könnte die Ertragsstabilität und damit die Produktivität der Linse deutlich verbessert werden. Diese Eigenschaften sind im Genpool der Linse nicht vorhanden. Die Pflanzenbiotechnologie bietet hier leistungsfähige Werkzeuge für das Erreichen dieser Zielsetzungen. Das Ziel der vorliegenden Arbeit ist die Etablierung eines reproduzierbaren und effizienten Transformationssystems für Lens culinaris Medik., welches die Integration von `Genes of interest´ (GOI) ermöglicht und somit die Grenzen der traditionellen Züchtung überwindet. Des weiteren soll ein markerfreies Transformationssystem entwickelt werden. Antibiotika- oder Herbizidresistenzgene sind für die Selektion transformierter Zellen und Gewebe Sehr nützlich, zur weiteren Kultivierung jedoch nicht unbedingt erforderlich. Die Elimierung der Markergene ist erstrebenswert, da es theoretisch nicht ausgeschlossen werden kann, dass Antibiotika- und Herbizidresistenzen auf pathogene Bakterien oder Wildkräuter übertragen werden könnten, obwohl die Wahrscheinlichkeit hierfür sehr gering ist. Aufgrund der Regenerationsfähigkeit wurde die Sorte Bari Musur (BM4) ausgewählt. Für die Transformation wurden dekapitierte Embryonen mit einzelnen Kotyledonen als Explantate eingesetzt. III Für die Transformation wurde der Agrobakterium Stamm EHA 105, mit dem Plasmid pSCP1D verwendet. Das Plasmid enthält sowohl das selektierbare Markergen bar , welches für die Phosphinothricin Acetyltransferase (PAT) kodiert, sowie ein pgip Gen der Himbeere ( Rubus idaeus L.), das das Polygalakturonase inhibierende Protein kodiert. Die Expression dieses rekombinanten Gens kann eine erhöhte Pilzresistenz gegenüber Pathogenen wie z.B. Colletotrichum und Botrytis bewirken. Die gesamte Kulturdauer von Aussaat der Samen und Transformation bis zur Gewinnung von transgenen Linsensamen betrug 2,5 bis 4 Monate. Dabei konnte eine Transformationseffizienz von etwa 29% festgestellt werden. Zur Entwicklung eines markerfreien Transformationssystems das bar Gen aus dem Plasmid eliminiert, so dass die T-DNA nur noch das pgip Gen enthielt. Aufgrund der ersten molekularen Analysen ergab sich eine Transformationsrate von 35%, diese Ergebnisse sollen in zukünftigen Analysen der Nachkommenschaften noch verifiziert werden. Auf funktionaler Ebene erfolgte die Analyse der Pflanzen durch einen semi-quantitativen Agarose Diffusionstest. Getestet wurde die Aktivität des pgip Gens gegenüber pilzlichen Polygalacturonasen von Colletotrichum lupini , C. acutatum und Botrytis cinerea . Das neu etablierte Transformationssystem könnte also eine effektive Möglichkeit zur Herstellung gentechnisch verbesserter markerfreier Linsen darstellen. Keywords: Agrobakterium , transgene, Linsen, PGIP, Pilzresistanz, markerfrei transformation. IV Table of Contents: ABSTRACT ...........................................................................................................................I ABBREVIATIONS .............................................................................................................IX List of figures.....................................................................................................................XI List of tables ................................................................................................................... XIII 1. INTRODUCTION......................................................................................................... 1 2. LITERATURE REVIEW 2.1 Legumes and Pulses - importance ....................................................................................4
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