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LINE 1. G ENETIC IMPROVEMENT AND CONSERVATION OF AGRICULTURAL CROPS AND FOREST SPECIES

The objective of this Research line is to increment, preserve, characterize, document, and place at the disposal of users the genetic diversity of species for current and potential use, preserved in the germplasm banks.

The research work is grouped under four sublines: Characterization Subline Its objective is to characterize, evaluate and document the phenotypic diversity of species that have agricultural, forestry, or microbial relevance, by using conventional and molecular tools. With the support of the U.S. Department of Agriculture (USDA), we have been able to characterize forest species such as mahogany (Swietenia spp) and chaperno (Lonchocarpus costaricensis), as well as the following agricultural species: peppers (Capsicum spp.), Cucurbitaceae (Cucurbita spp), and species of potential importance such as bitterwood tree (Quassia amara) and yam beans (Pachyrhizus spp). We have also studied the sapodilla (Pouteria sapota), cacao (Theobroma cacao), and introduced species such as coffee (Coffea spp.) and the Musaceae (Musa spp.).

Studies have begun recently on the diversity of phytopathogenic fungi, such as Mycosphaerella fijiensis, Moniliophthora roreri, and Mycena citricolor, which are agents that cause black sigatoka on Musaceae, moniliasis on cacao, and cocks eye disease on coffee. Despite the grave impact of these pathogens, many fundamental details are still unknown about their biology. Also, the characterization and appraisal of the genes in native coffee trees and the cultivated varieties of coffee (Coffea arabica) preserved in CATIEs germplasm bank. The coffee varieties grown in Latin America (Catuai, Caturra, Mondo Novo) originate from a very narrow genetic base, which greatly limits the possibilities of making genetic improvements. Nonetheless, CATIE possesses an important collection of coffee germplasm, including native genotypes. Along these lines, the Center, together with the Institute on Research for Development (IRD) from France, is conducting the characterization of certain

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Research aimed at the welfare of the peoples of the American Tr o p i c s

parameters of interest for regional genetic improvement, for instance, resistance to nematodes (Meloidogyne spp) and male sterility. At present, this work is under way using conventional and molecular methodology (AFLP). On the other hand, characterization has led to the evaluation of levels and dynamics of the genetic diversity of important tropical forest species. Such is the case of Mahogany (Swietenia macrophylla) and Cedar (Cedrela odorata), which are species with a great socioeconomic value; and genetic diversity studies have revealed that these are undergoing a very strong process of genetic erosion. Studies on the genetic diversity of Mayo Colorado (Vochysia ferruginea) and Piln (Hyeronima alchorneoides) an important Costa Rican species have evidenced the urgency in preserving the populations in the countrys southern and Atlantic regions, due to its high genetic variability. In turn, the characterization of beneficial and harmful microorganisms is gathering momentum, in view of the need to acquire more detailed knowledge on the diversity of organisms such as fungi and bacteria on agricultural and forestry activities. The Moniliophthora roreri fungus, which causes moniliasis in cacao, is one of the main factors hindering cacao cultivation

in Tropical America, causing losses that may reach up to 100% of production. Jointly with Reading University and CABI (both from United Kingdom), and with the financial support of USDA, its molecular characterization has led us to believe that this diseases country of origin is Colombia, where the broadest genetic diversity of this fungus has been identified. In cooperation with the International Center of Agricultural Research (CIRAD) and the International Network for Improvement of Banana and Plantain (INIBAP), we are conducting a research study aimed at describing the genetic structure of Mycosphaerella fijiensis populations, an agent that causes black sigatoka on banana and plantain in Latin America and the Caribbean. Preliminary results suggest that Honduras and Costa Rica possess the populations with the highest diversity indices. The data compiled shall prove very useful for future work on genetic improvement of these crops, improving combat strategies and quarantine measures, and focusing on the search of rustic genotypes with sources of resistance against these pathogens. Conservation Subline The objective of this subline is to develop strategies for the conservation of germplasm inside and outside the species natural habitat.

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It should be underscored that CATIEs plant collections of coffee, cacao, guava, tucuma (pejibaye), sweetsop, sapodilla, macadamia, annatto, pepper, and Cucurbitaceae, among others, are renowned both regionally and worldwide. For in situ conservation (at each species natural habitat), we are conducting studies on the impact of forest fragmentation on remnant populations of two species in the Dry Mesoamerican Pacific: Anacardium excelsum and Plumeria rubra. The outcome has shown the negative impact that this fragmentation has on the genetic base of A. excelsum, in the form of an endogamic depression on the fragmented populations as well as a reduced fertility. Genetic erosion illustrates that, in many cases, the population feasibility of A. excelsum and similar species cannot be guaranteed without providing added protection. The diagnosis and mapping of naturally-occurring mahogany populations (Swietenia macrophylla) from Mexico to Panama, proved that despite the fact that mahogany is grown in a broad spectrum of environments, its Mesoamerican populations have been severely thwarted, even in protected areas. In order to prevent genetic loss, it is imperative to establish efficient management and conservation programs in situ, ex situ or circa situ, thus guaranteeing its permanence.

As a part of the ex situ conservation activities, we created the largest live collection in the world of S. macrophylla, at CATIEs botanical garden. Moreover, a collection of Cedrela was planted, with over 300 accessions from throughout Mesoamerica, also preserved in CATIEs seed bank. This year the Plant Genetic Resources Unit placed emphasis on conservation, characterization and renewal of the collections of 776 accessions from 25 species. In order to preserve Musa, coffee, and cacao materials at the long term through cryoconservation, we have established protocols for the slow congealment of cellular suspension in six cultivars of Musa (Dominico, Currare, GE, GM, Col. 49, and SF 265). For the rapid cryoconservation of coffee seeds, we worked in collaboration with the IRD of France and the International Institute on Plant Genetic Resources (IPGRI). This technique is fast, simple, and inexpensive; it can be used in most of the regions countries which do not have the necessary equipment for slow cryoconservation. Also, this technique offers a great potential in forest species with recalcitrant seeds. During the year 2000 we successfully began cryoconservation of certain species of phytopathogenic fungi, among which Rosellinia and Mycosphaerella fijiensis stand out. This application is of utmost relevance in order to guarantee the storage of isolated fungi collected through time, and is very useful for the study of populations and development of disease-control strategies.

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Research aimed at the welfare of the peoples of the American Tr o p i c s

Propagation Subline Vegetative propagation is of vital importance for the multiplication of species from recalcitrant seeds with sterility problems, cloning of elite individuals, distribution of plantation materials, and conservation of ex situ germplasm. The objective of this subline is to promote and develop different strategies for propagation of: i) agricultural genetic resources, such as coffee, Musaceae, cacao, roots and tubers, among others; and ii) forestry resources, such as mahogany (Swietenia macrophylla), cedar (Cedrela odorata), and more recently, almond (Dipteryx panamensis), all of which was achieved with traditional and biotechnological tools. The large-scale micropropagation studies on coffee, conducted in cooperation with CIRAD, in support of the Regional Program on Genetic Improvement of Coffee (PROMECAFE), encompassed the introduction in vitro of F1 hybrids, the multiplication by somatic embryogenesis, and the distribution of hybrids and graftbearing varieties developed jointly with PROMECAFE. The reproduction of somatic embryos was optimized by using simplified bioreactors (RITA) with temporary immersion, followed by direct planting of the somatic embryos in a greenhouse.

In the year 2000 research on banana and plantain has been focused on achieving an optimum somatic embryogenesis in a liquid medium (embryogenetic cellular suspensions) and their application to the greatest number of cultivars, such as Gran Enano, Gros Michel, Williams, Currar, Dominico, FHIA1, FHIA 3, FHIA 23, and Lady Finger. Genetic improvement in Musaceae is very limited due to the different levels of ploidy and sterility in the most of the cultivars of commercial interest. We also worked on the development of methodologies for micropropagation of woody species, of great significance for multiplying and preserving recalcitrant species, threatened species, and elite individuals. Initial studies evaluated different cultivation media, concentrations and combinations of growth regulators to stimulate the sprouting of axilary gemmas of mahogany, cedar, and almond. At the same time, several trials have been conducted aimed at inducing somatic embryogenesis in mahogany. Improvement Subline This subline underscores work on priority agricultural and forestry crops, in response to the major biotic problems and augmenting the productivity of these species. As part of the activities, the project on Improvement of Resistance to Nematodes in Coffee (Coffea arabica L.) cultivars is currently under way, carried out in cooperation with the IRD (France).

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The attack by Meloidogyne spp. constitutes a severe problem in Central American coffee production; however, several sources of resistance and molecular markers have been identified for resistance to M. exigua. In addition, the research efforts undertaken clearly demonstrate that it is possible to transfer resistance genes to the C. arabica varieties, without affecting the quality of coffee. As far as banana and plantain cultivation is concerned, the most limiting disease in terms of production is black sigatoka (Mycospharaella fijensis), typically controlled by the intensive use of pesticides. Although in Musaceae the existence of genes which are resistant to the principal diseases are known, genetic improvement is extremely difficult, due to the high sterility index of cultivars. Current studies on genomics and molecular biology seek to identify resistance genes within the Musa genus, which will prove to be of great value in developing new strategies for the genetic improvement of banana and plantain crops for domestic consumption. On its part, the INCO Project Optimum Efficiency of New Genetic Improvement Strategies on Banana and Plantain for the Domestic Marketmanaged to obtain embryogenic calluses from different

diploid genotypes (Heva, T. la Gada, Colatino ouro, Chicame, Pisang madu, and Pisang jari buaya), and were able to establish embryogenic cellular suspensions of some of these. During this year, the selection activities of highly productive and disease-resistant cacao hybrids and clones, have allowed us to evaluate, under high pressure conditions of Moniliasis (M. roreri), the performance of 14 cacao clones, some of which were selected in Turrialba, Costa Rica, due to their tolerance to disease. After eight years of evaluations, we discovered that the EET-183 and CC-137 clones have an average production of 725 and 998 kg/ha/year, respectively, and an incidence of Moniliasis under 45%, as compared to internationally popular clones, such as Pound-7, UF-613, and Catongo, which recorded average productions under 178 kg/ha/year, and a disease incidence exceeding 83%. In the past five years, and with the financial support of the American Cocoa Research Institute (ACRI), five different experiments are under way in Turrialba and La Lola (Costa Rica), whose objective is selecting genotypes that offer high productivity and resistance to the main diseases in the area. These experiments will allow us to verify the behavior of these promissory materials, prior to beginning validation efforts with commercial producers.

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