Coffee (Coffea arabica cv. Rubi) seed germination: mechanisms and regulation

Coffee seeds display slow and variable germination which severely hampers the production of seedlings for planting in the following growth season. Little work has been done with the aim to understand the behavior of coffee seeds during germination and there is a lack of information concerning the regulation of the germination process. This thesis addresses questions concerning the mechanism and regulation of coffee seed germination.Initial experiments showed that radicle protrusion in the dark at 30 °C was initiated at around day 5 of imbibition. At day 10, 50% of the seed population displayed radicle protrusion and at day 15 most of the seeds had completed germination. The water uptake by the coffee seeds followed a common triphasic pattern as described for many other species (Chapter 2 and 3). During imbibition the coffee embryo grew inside the endosperm. The cotyledons increased in length by 35% and the axis by 40%, resulting in the appearance of a protuberance in the endosperm cap region. There was an increase in the embryo pressure potential up to day 5 of imbibition followed by a release of turgor thereafter, indicating relaxation of embryonic cell walls (Chapter 3). Light microscopy demonstrated that the cells of the embryonic axis displayed isodiametric growth (swelling) at the beginning of the imbibition process followed by both isodiametric and longitudinal growth later during imbibition. The isodiametric growth coincided with a random orientation of the microtubules whereas longitudinal growth was accompanied by a transversal orientation. Accumulation ofb-tubulin, an increase in the number of 4C nuclei and DNA replication were evident during imbibition. These cell cycle events coincided with the growth of the embryo and the appearance of cell division prior to radicle protrusion. However, cell division was not a pre-requisite for radicle protrusion in coffee seeds (Chapter 5).The endosperm of the coffee seeds possesses polygonal and rectangular cell types located in different parts of the endosperm. The endosperm cap cells have smaller and thinner cell walls than the rest of the endosperm, suggesting that the region where the radicle will protrude is predestined in coffee seed. Low temperature scanning microscopy revealed that during imbibition cells in the endosperm cap became compressed which was followed by a loss of cell integrity, appearance of a protuberance and occurrence of cell wall porosity (Chapter 3). As in many other species, the hemi-cellulose fraction of endosperm cell walls of coffee seeds consists mainly of mannans and galacto-mannans. These polysaccharides are commonly deposited in the cell walls as food reserve. Upon germination, these galacto-mannans are degraded through the action of hydrolytic enzymes, including endo-b-mannanase,b-mannosidase anda-galactosidase resulting in a weakening of the cell walls. The coffee endosperm cap weakens in two steps: cellulase activity correlated with the first step and endo-b-mannanase activity with the second step. Endo-b-mannanase activity appeared first in the endosperm cap and only later in the rest of the endosperm, and coincided with a decrease in the required puncture force and appearance of cell wall porosity. Different isoforms of endo-b-mannanase were found in the endosperm cap and in the rest of the endosperm. The activity ofb-mannosidase increased predominantly in the endosperm cap. However, low levels of endo-b-mannanase andb-mannosidase activities were also observed in the rest of the endosperm and in the embryo prior to radicle protrusion (Chapter 3 and 6). Two partial length cDNA clones encoding for endo-b-mannanase andb-mannosidase, respectively, were isolated from coffee endosperm caps. The deduced amino acid sequences exhibited high homology with those of other endo-b-mannanases andb-mannosidases from plants (Chapter 6). Abscisic acid (ABA) inhibited germination of coffee seeds but not their water uptake, isodiametric growth, increase in 4C nuclei and DNA synthesis in the embryo cells. In the endosperm cap ABA inhibited the second step of endosperm cap weakening, presumably by inhibiting the activities of at least two endo-b-mannanase isoforms. However, ABA had no effect on endo-b-mannanase activity in the rest of the endosperm or on cellulase activity. Two peaks of endogenous ABA occurred in the embryo cells during germination. The first peak was observed at day 2 of imbibition and the second (smaller) peak at day 5 of imbibition. The occurrence of these ABA peaks coincided with the increase in the embryo growth potential and the second step of endosperm cap weakening, which makes these processes possible targets of ABA action (Chapter 3).Exogenous gibberellin (GA 4+7 ) inhibited coffee seed germination. The response to GA 4+7 showed two sensitivity thresholds: a lower one between 0 and 1mM and a higher one between 10 and 100mM. However, it was shown that radicle protrusion of coffee seeds depended on de novo synthesis of GAs. Endogenous GAs were required for embryo cell elongation and the second step of endosperm cap weakening. Incubation of seeds in exogenous GA 4+7 resulted in a loss of embryo viability and the occurrence of dead cells, as observed by low temperature scanning microscopy. We suggest that the inhibition of germination by exogenous GAs is caused by factors that are released from the endosperm cap during or after its weakening. Exogenous GAs greatly accelerated the degradation of the endosperm cap. Factors that are involved in (normal) programmed cell death of the endosperm may reach the embryo during germination, causing cell death in the embryonic axis and, hence, inhibition of radicle protrusion. The results presented in this thesis show that coffee seed germination is controlled both by embryo growth and the second step of endosperm cap weakening (Chapter 4).Finally, the sequence of events during coffee seed germination and their interrelationships are presented and discussed (Chapter 7). The events occurring in embryo and endosperm all followed a two-phase pattern. The first phase occurred during the first 5 days of imbibition and the second phase thereafter, until radicle protrusion. The results make clear that the germination processes are temporally and spatially coordinated and that disturbance of this coordination, as in the presence of GAs, may severely affect seed behaviour