Αξιοποίηση μοριακών μεθόδων στη βελτίωση ανθεκτικότητας των εσπεριδοειδών στο Citrus exocortis viroid, στον έλεγχο της υγείας των εσπεριδοειδών υπό εξυγίανση και στη μελέτη διαφορικής έκφρασης γονιδίων ποικιλιών λεμονιάς

Abstract

Citrus, commercially grown in a wide range of soil and climatic conditions, aresubjected to substantial biotic and abiotic stresses which limit the production and insome cases, pose restrictions on the use of specific rootstocks and varieties. Citrusexocortis viroid (CEVd) causes severe symptoms in trees grafted on Poncirus trifoliata(L.) Raf. and its hybrids which, due to their tolerance to Citrus tristeza virus, arerecently employed for the replacement of sour orange (Citrus aurantium L.). The mosteffective method of controlling both biotic and abiotic stresses in plants refers to the useof resistant varieties. Nevertheless, the lack of natural genetic sources of resistance tomost severe diseases, along with the lack of basic knowledge on the inheritance patternof main agronomic traits, render necessary the use of genetic engineering in citrusbreeding. To this direction, the exploitation of genetic engineering requires theexistence of an efficient tissue culture protocol for each citrus species, to ensureregeneration of sufficient number of transformed plants. In this framework, the presentstudy initially focused on the determination of the most suitable genetic transformationprotocol for each citrus species: P. trifoliata, Carrizo citrange (C. sinensis × P.trifoliata), Citrumelo 1452 (P. trifoliata × C. paradisi), sour orange and “Maglini”lemon (Citrus limon (L.) Burm. f.) (Chapter 1).In recent years, one of the most efficient methods for the generation of resistanceagainst viruses and viroids refers to the exploitation of RNA silencing (RNAi). Towardsthis direction, and in view of the recalcitrant nature of citrus species which renderstransformation and regeneration particularly difficult, aim of this study was thedevelopment of transgenic CEVd resistance in the model plant Nicotiana benthamianawhich is a non-host of CEVd. In this line, the CEVd-inoculation of N. benthamianaplants was pursued by two different approaches: a) agroinfiltration and b) stable genetictransformation, using a plasmid harboring the dimeric CEVd molecule (Chapter 2). Theresults indicate a low rate of CEVd replication in the agro-infiltrated plants, in contrastto transgenic plants which were capable of CEVd replication. The latter though, werecharacterized by reduced growth and seed production compared to wild-type plants. Towards investigating the capability of various segments of the CEVd genome ininducing silencing of its genome and subsequent suppression of its replication,transgenic plants replicating the viroid were inoculated with two different selfcomplementaryhairpin RNA fragments from the CEVd genome (Chapter 3). Theresults provided strong evidence that both CEVd regions are capable of triggering RNAsilencing, thus causing a reduction to the viroid replication rate. These results were alsoconfirmed by transient expression experiments in wild-type N. benthamiana plants,where viroid presence caused reduced accumulation of the selected CEVd fragments.As a means to investigate whether the observed resistant phenotype of the modelplant N. benthamiana can be achieved in citrus species, which consist the natural viroidhosts, the introgression of two selected segments of the CEVd genome was pursued, bygenetic transformation, in various citrus genotypes (Chapter 4). To this purpose, genetictransformation was performed in plants of P. trifoliata, Carrizo citrange and Citrumelo1452. Parallel aim was the generation of transgenic resistance against citrus psorosisdisease, through the introgression of the viral coat protein of Citrus psorosis virus(CPsV) in sour orange and “Maglini” lemon (Chapter 4).The most important method to control citrus diseases is the use of healthy certifiedpropagation material. An effective method for plant sanitation is in vitro micrograftingof apical meristems. The study of in vitro micrografted lemon, orange and mandarinplants verified the absence of the most important viruses and viroids for Greekcitriculture, indicating that the necessary expertise for sanitation of the precious citruspropagation material exists in our country (Chapter 5).Finally, this dissertation included the study of certain sequences which areoverexpressed in the Greek lemon ”Adamopoulou” compared to the Portuguese”Lisbon” (Chapter 6). The difference between the two varieties is that ”Adamopoulou”is tolerant to mal secco and cold compared to “Lisbon”. Several of the sequences understudy presented homology with proteins directly or indirectly involved in the defensemechanisms of plants against biotic and abiotic stresses, with the difference between thetwo varieties in expression of seven genes being at varying levels.