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First Cryopreservation Seed Bank For Arabica Coffee

September 14, 2000

The storage of coffee (Coffea arabica) seeds to ensure preservation of their genetic resources is an important problem. Like many other tropical plants, coffee seeds cannot be conserved in cold storage because they remain cold-sensitive. IRD (Institut de recherche pour le développement), with the financial backing of the BRG (Bureau des ressources génétiques, Paris) and the IPGRI (International Plant Genetic Resources Institute, Rome) launched a research programme in 1996 on seed preservation techniques. The objective was to devise a routine method for long-term conservation of Coffea arabica seeds. Researchers at Montpellier have developed a cryopreservation procedure, entailing freezing in liquid nitrogen at -196°C, for this species. The method has recently been applied at the CATIE (Centro Agronomico Tropical de Investigation y Ensen'£nza in Costa Rica) on about 100 genotypes of the species representative of the genetic diversity of the collection of coffee trees grown at that research institute. The method so elaborated could be used to establish the first C. arabica germplasm cryobank in the world.

Nearly 6 million tonnes of coffee are produced each year in tropical countries. This puts coffee beans among the prime agricultural products exchanged in the world. Although there are about 100 species of coffee tree, the bulk of the production of coffee for drinking rests on just two of them: Coffea arabica and Coffea canephora, alias Robusta. Beans from C. arabica contain two to three times less caffeine than those of C. canephora, are less bitter and have more flavour. They make up two-thirds of the world's coffee production.

The seeds of most plants withstand desiccation and low temperatures. Known as "orthodox" seeds, they can therefore be stored and preserved in cold storage for several decades. The genetic resources of many species with orthodox seeds are conserved in this way. Collections of millet, rice and sorghum are thus stocked in cold rooms at IRD's Montpellier centre. However, many tropical species with strong economic potential like coffee, oil palm, coconut or hevea, are an exception because their seeds prove to be highly sensitive to desiccation or to cold or to both. These are so-called recalcitrant seeds. Moreover, under ambient temperatures and relative humidity, coffee grains cannot be kept more than a few months.

Storage of C. arabica seeds has always been a major problem hindering attempts to preserve its genetic resources. Currently, these are essentially kept in cultivated "field" collections, a preservation method fraught with disadvantages. The plantations cannot be protected from the vagaries of the weather, diseases or attack by pests. In addition, some genotypes adapt with difficulty to the environment to which they have been transplanted. Furthermore, maintenance of these "field" collections requires an enormous amount of time and space and is therefore costly. Coffee seedlings can also be preserved using in vitro culture of microcuttings. A number of problems are attached to this method, however, such as substantial technical constraints and a regular maintenance of the resulting "vitroplants", making it expensive.

These reasons prompted IRD to undertake research on alternative methods of preservation of the genetic resources of coffee seeds and, notably, on cryopreservation (preservation in liquid nitrogen, at -196°C). These studies have been conducted jointly with CATIE with financial backing from BRG and IPGRI.

Seeds of the various species of coffee react differently to desiccation and immersion in liquid nitrogen. Their resistance to cryopreservation must therefore be studied case by case. Coffea arabica, whose seeds do not survive direct immersion in liquid nitrogen, needs extensive research. In the same seed the embryo and the reserve tissue (endosperm), which make up 99% of the seed, have different degrees of cold sensitivity. The first tolerates low temperatures whereas the second is highly cold-sensitive. The conditions therefore had to be defined that would allow both the embryo and the reserve tissue to survive.

At the end of several years of research on the plant material supplied by CATIE, which has at its disposal one of the most comprehensive coffee tree collections in the world, a cryopreservation procedure has been devised for Coffea arabica. In four steps, each requiring specific values of the parameters involved: desiccation, freezing, rewarming and rehydration of seeds. The seeds must be desiccated to a very precise point (0.2 g of water per gram of dry weight) obtained following three weeks' equilibration under 78% RH. Freezing must be done in two stages, vital for successful cryopreservation. This means a slow-cooling step at 1°C per minute down to -50°C, followed by rapid immersion in liquid nitrogen. Rapid rewarming, thawing, is carried out before controlled rehydration, which is obtained by seed culture on low-osmotic potential solutions, in other words in an aqueous solution which allows rehydration to proceed more slowly. The researchers have thus shown for the first time that this technique, called "osmopriming", or osmoconditioning, used mainly by seed manufacturers to improve seed viability, can also have a considerable beneficial effect on cryopreservation. If all these conditions are fulfilled, the cryopreserved C. arabica seeds have a germinating power equivalent to that of the normal, control seeds.

This cryopreservation protocol is a straightforward, effective and inexpensive method for genebanks which cannot afford in vitro culture techniques. Using this approach many high-volume batches of seeds can be treated simultaneously. The method's success is a pointer to future applications to other species of the Coffea genus, whose germination power is seriously impaired by cryopreservation.



Institut de Recherche pour le Développement, Paris (IRD)