Effect of cold storage, heat, smoke and charcoal on breaking seed dormancy of Arctostaphylos pungens HBK (Ericaceae)

Resumen. En este estudio se evaluó el efecto del almacenamiento en frío y factores relacionados con el fuego en la germinación de Arctostaphylos pungens HBK (Manzanita Mexicana), un arbusto común en bosques de pino-encino mal manejados en Durango, México. Debido a que este arbusto presenta una alta densidad en bosques que han sido incendiados, investigamos el efecto de alta temperaturas, humo y carbón sobre la germinación de las semillas. Se trabajó con semillas recién colectadas de los arbustos. El porcentaje de germinación más alto que se obtuvo fue de 30%, para semillas que se almacenaron en condiciones frías, con tratamiento de shock térmico, pasadas por humo y regadas con dilución de carbón vegetal.


INTRODUCTION
In ecosystems adapted to fire, two main strategies can be found for plant reproduction.These strategies include heat resisting (often underground) structures, and heat resistant seeds (Keeley, 1991).Arctostaphylos is a genus with 150 species, many of which inhabit fire-prone ecosystems like chaparrals, and cold and temperate forests (Berg, 1974;Weise et al., 1991).Species in this genus can survive fire as adult plants or germinate after fire (Weise et al., 2005).
Seeds of Arctostaphylos resemble a small nut from 1 to 3 mm length with 3 to 7 seeds and a hard endocarp.While the mesocarp is edible, the exocarp is a thin paper-like structure.Seeds are water permeable; however, seed germination of this genus is not clearly understood, since it does not germinate in contact with water (Bliss, 1958;Berg, 1974).Seeds from Arctostaphylos pungens have none or very low germination (Martínez-Pérez et al., 2006) even after applying conventional treatments such as mechanical scarification, cold storage and different light regimes, and using seeds either from different ages or collected from dung of dispersal agents (Márquez-Linares, 2004).Perhaps variables associated with fire (such as high temperature and smoke) break seed dormancy.However, no studies have investigated the effect of fire on the germination of Mexican Manzanita.We explored whether germination of Arctostaphylos pungens might be induced by cold storage, heat, smoke and charcoal.This would allow to better understand field conditions that might result on the encroachment of this shrub in Durango forests and chaparrals.

MATERIALS AND METHODS
Freshly matured seeds of Arctostaphylos pungens were collected from randomly selected plants (n=20) on June 2003 at 2440 m above sea level.Seeds were cleaned in the lab from fruit remains.Seeds that (1) were malformed, (2) had insect perforations, and (3) were not easy to separate were discarded.Clean selected seeds were placed in water for one minute, and those that floated were considered unviable and discarded.A set of seeds was tested for viability using the tetrazolium test (Sutherland, 1998).Extra seeds were added so the number used for germination included at least 50 viable seeds per replicate.
Seeds were placed inside Petri dishes with filter paper.Cotton wool was placed under the filter paper to keep moisture and prevent excess available water.Seeds were positioned with the hypocotyl facing downward, in contact with the moist filter paper.Petri dishes were plastic sealed to keep moisture, and incubated at 20 °C.An emerging radicle was the indicator of a germinated seed.Germination was registered every three days for eight weeks, removing germinated seeds.
We tested the effect of (a) Heat at 100 °C and 120 °C: seeds were placed during five minutes inside a muffle furnace with an electronic temperature control.Temperature was set one degree higher than that reported for each trial to allow for cooling while opening the door.Temperature was independently registered (± 1 °C) using a thermometer with the bulb on the top of the seed tray; (b) Cold storage, by placing seeds 40 days at 4 °C; (c) Smoke: leaves and twigs of Mexican Manzanita were charred on a hot metal plate until they ignited without flame.Smoke was captured using an extractor, and directed toward seeds placed on a wire mesh for 5 minutes.Smoke temperature was < 38 °C when reaching the seeds.In this way, smoke effect was not confounded with heat effect, and (d) Smoke residues, where seeds were watered with burnt Mexican Manzanita residues following Keeley and Fotheringham (1998).These residues were obtained after twigs and leaves of Mexican Manzanita were charred on a hot metal plate.Residues were placed in a beaker with distilled water and stirred.After 12 h, this material was filtered using a 2 mm mesh and diluted to 5%.This dilution was used to water seeds inside the Petri dishes.A control group (e) of seeds kept at room temperature was also tested for germination.Four Petri dishes with 50 viable seeds each were used as replicates for each treatment.
Statistical analysis.Data were analyzed using a four-way ANOVA.Adjusting for viability, germination results were converted first to percentage, and then arc-sine transformed before ANOVA (GLM, SAS, 1989).

DISCUSSION
Seed germination of A. pungens was promoted by cold storage suggesting an after winter effect on seed germination.This is also the case in other species in temperate environments (Baskin & Baskin, 1998;Jurado & Flores, 2005).Seed germination was higher when using fire related stimuli: e.g., the combination of high temperature, presence of smoke and watering with charcoal.Post-fire germination fire is in agreement with other species in the genus for California.Keeley (1987) found that lixiviates of charred wood promoted seed germination of several California Chaparral species, including A. glandulosa and A. patula.He argued that germination is promoted by the presence of an oligosaccharide that results from heating hemicelluloses during mild fires; however; it has recently been found that germination is promoted by active butenolide present in plant-derived smoke (Flematti et al., 2004;Merritt et al., 2006;Daws et al., 2007;Stevens et al., 2007).Parker & Kelly (1989) found that seeds of A. canescens extracted from the soil germinated when watered with charred wood extract.The effect of smoke promoting germination has not been determined for Arctostaphylos, but it has been shown to promote seed permeability in several species of the Californian Chaparral (Keeley & Fotheringham, 1998).Heat did not stimulate germination by itself either for A. viscida and A. mewukka (Kauffman, 1986;Kauffman & Martin, 1991) or for A. patula and A. glandulosa (Keeley, 1987).
We found that several factors combined increased germination percentage of Mexican Manzanita seeds.This suggests a synergic effect in the combination of variables such as cold storage, smoke, and heat and charcoal.However, germination was relatively low (ca.30%) even with a combination of cold storage and fire-simulating variables.It is possible that other factors such as fluctuating light regimes (Baskin & Baskin, 1998;Pons, 2000;Flores et al., 2006) might promote germination.However, it is also possible that only very few seeds of Mexican Manzanita germinate each year after winter, like other temperate climate species that form a seed bank ( Jurado & Flores, 2005;Ibáñez et al., 2007).However, after a fire many seeds would germinate, like those from fire-adapted species (Álvarez et al., 2007;Luna et al., 2007;Paula & Pausas, 2008).The cold storage conditions, and the conditions made to simulate fire such as heat, smoke and charcoal used here, might have been only similar to natural winter temperature variations in the field, and the effects of a real fire.However, they were sufficient to trigger germination on a relatively small percentage of seeds.Further experiments manipulating the variables tested here, and others, might help to determine the natural conditions that trigger establishment of large numbers of Mexican Manzanita seedlings in Durango Forests and Chaparrals.Tabla 2. Análisis de Varianza de la germinación de semillas de Arctostaphylos pungens HBK (Manzanita Mexicana) de una combinación de tratamientos.

Table 2 .
Analysis of Variance on seed germination of Arctostaphylos pungens HBK (Mexican Manzanita) from a combination of treatments.