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Seed pretreatment methods to improve germination of the multipurpose West African forest species Dialium guineense
Forest Ecology and Management, 57 ( 1993 ) 257-273
257
Elsevier Science Publishers B.V., Amsterdam
Seed pretreatment methods to improve germination of the multipurpose West African forest species Dialium guineense A.H. Todd-Bockarie and M.L. Duryea University of Florida, Department QfForestry, Gainesville, FL 3261 I, USA (Accepted 7 May 1992 )
ABSTRACT Todd-Bockarie, A.H. and Duryea, M.L., 1993. Seed pretreatment methods to improve germination of the multipurpose West African forest species Dialium guineense. For. Ecol. Manage., 57: 257273. Two experiments preceded by a pilot study were conducted to determine the optimal method for overcoming seed coat dormancy of Dialium guineense Willd. Seeds from parent trees were kept separate to determine whether genetic variation in dormancy influenced pretreatment choice. There were pronounced differences between parent trees in germination response for the control and the two lower-germinating pretreatments, indicating that differential dormancy exits within this species. Nicking and concentrated sulfuric acid pretreatments effectively improved the germination response for seeds from both dormant and non-dormant parent trees. Sulfuric acid pretreatments are recommended for well-equipped centralized nurseries, whereas nicking is most appropriate for small village nurseries.
INTRODUCTION
Dialium guineense Willd., or velvet tamarind, is a multipurpose tree of the West African Guinean Forest zone. It typically grows in dense savanna and riparian forests, ranging from Senegal to the Sudan, along the southern boundary of the Sahel (Szolnoki, 1985). It is a leguminous species and belongs to the Caesalpiniaceae family. The velvety-black fruit of Dialium guineense contains from one to five hard seeds surrounded by a bright orangered pulp. Fruits ripen in April to May during the dry season (Savill and Fox, 1968 ). Elephants, birds, monkeys and humans disperse the seeds when they eat the acid-tasting astringent fruit pulp (Dalziel, 1948; Irvine, 1952 ). At least three primate species (Spot-nosed monkey (Cercopithecus nictitans), CampCorrespondence to."A.H. Todd-Bockarie, University of Florida, Department of Foresty, Gainesville, FL 32611, USA.
© 1993 Elsevier Science Publishers B.V. All rights reserved 0378-1127/93/$06.00
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A.H. TODD-BOCKARIEAND M.L. DURYEA
bell's monkey (Cercopithecus campbelli) and the Chimpanzee (Pan troglodytes) ) frequently feed on Dialium fruit (M. Conteh, personal communication, 1989).
Dialium guineense serves two important functions for the West African farming family. First, it is an energy-efficient and preferred charcoal species for rural villagers who depend on wood for all their energy needs (Kamara, 1986; Cline-Cole, 1987 ). Second, the fruits and leaves are consumed in critical food shortage periods during the dry season when farm produce is unavailable (Okafor, 1978; Falconer and Koppell, 1990). In addition to these two primary functions, the tree provides a variety of different products throughout the cropping calendar (Table 1 ). Given the pressing need for increased food production in many West African nations, improvements in tree crop production methods for this species are needed. Under minimal tillage swidden agricultural operations, the tree regenerates naturally during the fallow period (Nyerges, 1989). Currently, farmers wait for volunteer seedlings to produce harvestable fruits in farm fallows (Okafor, 1980b). However, as pressure on available land resources inTABLE 1 Multiple uses of Dialium guineense by West African farm families Part
Description of use
Reference
Fruit
Edible fruits rich in Vitamin C A thirst-quenching beverage Processed into jams and jellies Income supplement for women
Okafor, 1978 Okafor and Fernandes, 1987 Okafor, 1980a Kamara, 1986
Leaves
Vegetable (fermented for flavor) Fodder for livestock Medicine for diarrhea Mulch Eye ointment
Szolnoki, 1985 Okigbo, 1976 Ayensu, 1978 Von Carlowitz, 1986 Dalziel, 1948
Wood
Charcoal Fuelwood Resistant to termites Poles and bridge construction Carvings, utensils and tools
Cline-Cole, 1987 Kamara, 1986 Gotz, 1983 Gotz, 1983 Okigbo, 1976
Bark
Mouthwash and toothache cure
Ayensu, 1978
Branches
Toothbrushes or chewsticks Stakes for trailing yams Hedgerow and windbreak
Dalziel, 1948 Okigbo, 1976 Von Carlowitz, 1986
Roots
Erosion control
Nyoko, 1980
METHODS TO IMPROVE GERMINATION OF DL4LIUM GUINEENSE
259
creases and fallow periods shorten, secondary succession for tree species is inhibited and grasses and forbs are favored (Unesco/UNEP/FAO, 1978; Nyerges, 1987; Longman and Jenik, 1987 ). The lack of information on appropriate regeneration methods limits the use of this species in reforestation activities. Regeneration of Dialium from seed is difficult because seed coat dormancy inhibits germination. Poor germination may be linked to the rupturing of the distinctive waxy cap in the hilar region, which is directly connected to the fiat, disk-shaped embryo in the interior of the seed. Seed pretreatment is needed for quick, uniform germination. A range of different seed pretreatment methods have been successfully used in rupturing the water-impermeable testa of other tree species to overcome dormancy (Rolston, 1978; Werker, 1980). Chemical pretreatments such as immersion for several minutes in concentrated sulfuric acid have been particularly effective in overcoming physical dormancy in other leguminous species (Von Carlowitz, 1986). However, laboratory grade acids are expensive, difficult to obtain and pose a safety problem when used in village nurseries (Willan, 1985). In West Africa there are several alternative pretreatment methods which may be both effective in breaking seed coat dormancy and widely available in the rural villages. Strong acids found in the digestive tract of domestic livestock are one possibility. Ruminants have been employed as a pretreatment for hard seed with varying levels of success in several countries including Senegal for Adansonia digitata L. (Von Maydell, 1983 ), Kenya for Acacia tortilis (Forsskal) Hayne. (Kamveti, 1982), and Pakistan for Cassia fistula L. (Sheikh, 1980). By varying immersion time and temperature, hot water treatments have effectively been used to break seed coat dormancy in several related legumes (Werker, 1980). Mechanical pretreatments such as nicking, sanding, aging, threshing and burning have also improved percentage germination in many hard-seeded species (Msanga and Maghembe, 1986; Kariuki and Powell, 1988; Duguma et al., 1988; West and Marousky, 1989). Genetic variation in seed characteristics is commonly exploited by plant breeders to create high-yielding cultivars for very specific purposes. For example, geneticists have effectively increased both the protein and the oil content of soybean seeds to produce a more nutritional product for the market (Shannon et al., 1972 ). Similarly, a genetic basis for seed coat impermeability evident in many agronomic species allows plant breeders to reduce the proportion of hard seed in a hybrid to promote quick, uniform germination (Rolston, 1978). The inter-relationship between environmental and genetic factors in controlling variation in seed coat permeability is not fully understood. However, several authors have noted distinct differences among seed sources of hard-seeded leguminous tree species to pretreatments for overcoming dormancy which may imply some genetic basis for impermeability ( Don-
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A.H. TODD-BOCKARIE AND M.L. DURYEA
nelly et al., 1972; Gupta et al., 1973; Olvera and West, 1985 ). Given that over 85% of legume species possess the hardseededness trait (Rolston, 1978 ), manipulation of seed coat permeability by breeders would have a significant impact on nursery practices by decreasing or eliminating the need for pretreatments. The objectives of the present study were two-fold. First, a wide range of pretreatment methods appropriate for village nurseries in West Africa were tested to establish the optimum germination response. Second, we sought to determine whether genetic variability in seed coat dormancy existed for the species, and ascertain what effect this may have on pretreatment choice. METHODS
Seed collection Seed was collected from 10 open-pollinated Dialium guineense trees at the close of the dry season in March 1989 by Peace Corps Volunteers, researchers and Tiwai Island Wildlife Sanctuary staff. The collection area was located in Barri and Koya Chiefdoms, Sierra Leone (45 km inland from the Atlantic Ocean in the southeastern forest region; latitude 7°N, longitude 7 °W). A minimum distance of I km was maintained between trees so that each collection would represent a separate genetic line. Fruits were cleaned and seeds with obvious insect and fungal infestations were removed. After a 7-day drying period, seeds were packaged separately by parent tree and shipped by air to the University of Florida, Gainesville (USDA Permit 37-72360). All seed was stored in sealed silica-gel-lined containers in an air-conditioned room at 18 ° C until being used in the experiments.
Germination All germination tests were conducted using a Percival 1-30B plant growth chamber under a controlled fluorescent light and a temperature regime of 25 ° C, with a 12 h day/night cycle, to approximate growing conditions in Sierra Leone. A series of pilot studies and two experiments were conducted to test 39 pretreatments (Table 2). A bulk seedlot, which was a mixture of seeds from five different parent trees (Nos. 6-10), was used in Experiment 1. A second group of five different parent trees (Nos. 1-5 ) was tested separately in Experiment 2. The most promising methods were retained from the pilot studies for the experiments. We blocked by shelf in the germination chamber to minimize differences due to gradients in humidity and temperature (Lee and Rawlings, 1982 ). Seeds from each parent tree were surface-sterilized for 5 rain in a 20% chlorine solution prior to treatment. Treated seeds were sown in 9 mm petri plates lined with 2 layers of filter paper. Each plate was sealed
METHODS TO IMPROVE GERMINATION OF DIALIUM GUINEENSE
261
TABLE 2 Thirty-nine seed pretreatment m e t h o d s employed during the pilot study and Experiments 1 and 2 Number'
Pretreatment description
Pilot
Experiment
1
Control
x
x
Chemical 2, 3, 4 5, 6, 7, 8 9, 10, 11
95% ethanol: 1, 3 or 6 h H2SO4: 1, 7, 8 or 10 min H2SO4: 2, 4 or 6 min
x
Heal 12 13,14,15 16,17,18 19,20,21 22 23,24,25 26 27,28,29,30
Warm H20:5 min at 80°C Warm H20: 2, 4 or 6 min at 80°C Boiling H20: l, 2 or 3 min at 100 ° C Shock2: l, 2 or 3 min at 1 0 0 ° C / 3 4 ° C Cold H20 soak: 48 h at 25 °C Hot H20 soak: 6, 8 or 24 h at 100°C Dry heat: 15 min at 100°C Fire: 0.5, 1, 1.5 or 7 min at 2400°C
x
x x
x x x x x x x x
Meehanical 31 32, 33 34, 35
Nicking Commercial mill: 1 or 2 min Coffee grinder: 1 o r 2 s
x x
Combination 36 37, 38, 39
Sheep ingestion Accelerated aging3: 2, 4 or 6 days
x x
x
x
x x
~Consecutive numbers on a line represent several pretreatment combinations. 2Shock was applied by immersing the seeds in boiling water followed by ice water for the specified time period. 3Accelerated aging consisted o f placing seeds in a controlled environment o f 100% relative humidity over a water bath maintained at 40 °C for several days.
with Parafilm after 10 ml of distilled water had been added. Germination, defined as the emergence of the radicle beyond 2 mm, was recorded daily for a 14-day period.
Tetrazolium chloride test procedure At the close of each germination experiment, the viability o f all ungerminated seeds were assessed using a tetrazolium chloride test. The testa of each seed was nicked with a scalpel before being allowed to fully imbibe distilled water over a 24 h period at 20 ° C following the methodology of Moore ( 1985 ). The seed coat was then removed from the imbibed seed and a longitudinal
262
A.H. TODD-BOCKARIEAND M.L. DURYEA
TABLE 3 Topographical stain evaluation classes (Moore, 1985 ) Class
Description
Viability
1 2 3 4 5
Uniform red staining of embryo and radicle Pale pink staining of embryo and radicle Cotyledons < 50% unstained Radicle unstained or damaged No staining
Germinable Germinable Weak germination Non-germinable Non-germinable
section through the endosperm was cut to allow the embryo tissue to imbibe the stain. Prepared seeds were immersed in a 1% solution of 2,3,5-triphenyl tetrazolium chloride for 9 h in the dark at 35 °C. The topographical staining pattern of the embryo, cotyledons, radicle and endosperm was evaluated for each seed and used to place the seed into one of five viability classes (Moore, 1985 ) (Table 3 ). Percentage live seed for each treatment X parent tree combination was calculated by first adding together the number of seeds in the three germinable evaluation classes ( 1-3 ) and then dividing this sum by the total number of seeds.
Pilot studies A series of non-replicated pilot studies were conducted on a bulk seed lot to initially test and eliminate pretreatment methods which did not alter seed coat dormancy. Pretreatment methods were grouped into four categories as follows: (1)chemical; ( 2 ) h e a t ; (3)mechanical; (4)combinations which consisted of two or more types of treatments to the seed coat (Table 2 ). Separate 50-seed samples were used in testing a range of treatment combinations within each category. A total of 14 pretreatment combinations were screened in order to find the most promising treatment combinations in improving percentage germination.
Experiment I Using a bulk seedlot which was a mixture of seeds from five parent trees (Nos. 6-10), 25 pretreatment combinations were tested in Experiment 1 (Table 2). The most promising treatments from the previous pilot studies were used. A randomized complete block design was employed with 25 pretreatments blocked by shelf in the growth chamber (1500 total seeds= 4 blocks X 25 pretreatments X 15 seeds).
METHODS TO IMPROVE GERMINATION OF DIA LIUM G UINEENSE
263
Experiment 2 The four pretreatment combinations with the best percentage germination together with a control were selected for testing with parent trees (Nos. 1-5 ) in Experiment 2 (Table 2). A randomized complete block design was employed with four blocks, five parent trees and four pretreatments: ( 1 ) nicking; (2) 8 min sulfuric acid immersion; (3) 8 h soaking in hot water; (4) 1 s grating in a coffee grinder (1500 total seeds = 4 blocks × 5 pretreatments X 5 parent trees × 15 seeds). Imbibition measurements and scanning electron microscopy were utilized to characterize parent tree X pretreatment interactions.
Imbibition measurements Water uptake was measured for two seeds sampled randomly from each of the five parent trees and five treatments in all four blocks in Experiment 2. Samples were taken during the imbibition (Phase A) and activation (Phase B) stages of germination. The five time intervals for sampling were ( 1 ) control prior to treatment, and (2) 0 h, (3) 8 h, (4) 16 h and (5) 24 h after treatment. The fresh weight of each seed sample was measured using a Sartorius H-51 analytical balance. After being dried for 48 h at 70°C, each sample was then reweighed. The percent water absorption was calculated for all samples based on the initial moisture content of the seed following the method of Saha and Takahashi ( 1981 ).
Scanning electron microscopy A sample of four seeds was randomly selected from every parent tree in each of the five treatments in Experiment 2 and dried in a vacuum-sealed desiccator before mounting on a l u m i n u m stubs. Each seed was sputter coated with gold for 6 min before being viewed in a Hitachi 450-S scanning electron microscope at an accelerating potential of 20 kV. Disruption of the strophiole, hilum, micropyle and testa were recorded for each seed. Representative seed samples were photographed for each treatment.
Statistical analys& An analysis of variance (ANOVA) was conducted on percentage germination for all germination tests. The data were transformed and arcsin values were used in performing the analysis (Snedecor and Cochran, 1967). Duncan's multiple range test was used to test the significance of general treatment and parent tree effects, and single degree of freedom contrasts were used to determine the significance between specific treatment and parent tree comparisons. All treatments were considered statistically significant at P = 0.01.
264
A.H. TODD-BOCKARIE AND M.L. DURYEA
Treatment and parent tree differences in water absorption rates for the imbibition measurements were analyzed using an ANOVA at each time interval. An additional ANOVA was performed on water absorption combined across all five time periods because the mean square error (MSE) for all intervals were comparable; time period was treated as a sub-plot factor in a split-plot design. RESULTS
Pilot studies Nicking the seed coat surface with a scalpel produced the highest percentage germination (96%) compared with the control (10%). The three most promising treatments were the coffee grinder, sulfuric acid and water soak pretreatments. Substantially higher percentage germination resulted from 2 s of grinding (65%) compared with only 1 s (10%). Immersion for 7 min in concentrated sulfuric acid (55%) was the next most promising pretreatment, with the 1 min immersion (25%) and the 10 min immersion (4%) producing considerably lower germination values. Temperature appeared to affect the response to water soak pretreatments. A quick 5 min immersion in water at 80 °C (32%) was more effective than a longer cold water soak at 25 °C (8%). Extreme heat pretreatments, such as 7 min fire and 15 min dry heat ( 100 ° C), killed the seeds. Employing the digestive tract of sheep as a pretreatment was equally ineffective; all the seeds were digested.
Experiment 1 Five of the 25 pretreatments produced encouraging germination responses, including nicking, sulfuric acid immersion and water soak (Fig. 1 ). The nicking and sulfuric acid pretreatments were significantly better than the remaining treatments (P=0.001) or the control (P=0.001). Nicking produced the best germination response overall, and was significantly higher than any of the sulfuric acid treatments ( P = 0.001 ). The 6 h water soak resulted in 22% germination, which was not statistically different from the control (10%). However, it was viewed as promising if further refined. Twenty of the 25 pretreatments resulted in germination responses equal to, or lower than, the control. The tetrazolium test indicated that the extreme heat treatments killed a significant proportion of the seed ( P = 0.001 ) (Fig. 2 ). As illustrated by the fire, boiling water and shock pretreatments, the longer the exposure time to heat, the greater the proportion of dead seed. In contrast, an equal proportion of the seed remained alive when compared with the control for the aging, ethanol soak, and warm water treatments, although the combinations used did not overcome dormancy.
METHODS TO IMPROVEGERMINATIONOF DIALIUM GUINEENSE
265
100 95
90 85
a
80 75 70
._o 65 60 55 50 45
u
b
b
40 35 30
25
¢
0
Seed Pretreatment
Fig. 1. Mean percentage germination for the twelve most promising seed pretreatment methods compared with the control for Experiment 1. Pretreatments with the same letter were not significantly different. 100 90 95% LSD I 80
7O 60 "0 a) 50
Or) Q
_> 40 ,-I
30 20 10 0
.o j . ~ o ~ . ~ o ~ - o
,, ~o-,~ ~ , , ~ , , ~ : ~ , , ~ ,
~ ~. ~ ~
Treatment
Fig. 2. Mean percentages o f live seed by p r e t r e a t m e n t c o m p a r e d with the control for the tetrazolium chloride test in Experiment I.
266
A.H. TODD-BOCKARIE
90 10070
A N D M.L. D U R Y E A
Tree 1
o~60
Tree 2
C O
Tree 3
~60
U,
Q
Tree 4
I Tree 5
.... • ....
• 5o
I--
30
b"
e= 20 n 10 0O
I 20 3040
li0
~)
60
70
60
9~ ,100
CONTROL 1120 GRIND
Pretreatment
NICK 112SO4
Germination
(%)
Fig. 3. Differences in mean percentage germination among five parent trees for four pretreatment methods compared with the control in Experiment 2.
100 95% LSD = CONTROL "
90
I
NICK --"@o-
/
80
GRIND ....G....
//
HZS04
70
!
I
//
/
/
A V
H20 SOAK
60
--A.-.
/
~
/
//
/
IN/
~. so
/
/
E
/ /
,
/
/
/
t
i
/:' /:" t-Y ~:.
,&. . . . . . . . . . . . . .
•
30
/
/
/
20
/ /
/
/ / /
k"" ..--
.oO°°
10
HR
I
8 HR Time
I
16 HR
I 24 HR
(Hours)
Fig. 4. A comparison among water uptake patterns for the control and four pretreatment methods during the first 24 h of imbibition in Experiment 2.
Experiment 2 A satisfactory mean germination response was obtained for the nicking (90%) and sulfuric acid (93%) pretreatments compared with the control
METHODS TO IMPROVE GERMINATION OF DIALIUM GUINEENSE
267
(29%), 1 s grating in a coffee grinder (50%), or 8 h water soak (36%). There were pronounced differences between parent trees in germination response for the control and the two lowest-germinating pretreatments (Fig. 3 ). A separate ANOVA was used to compare the germination responses of different parent trees within the control and the two lowest-germinating pretreatments. Seeds from parent trees Nos. 4 and 5 were significantly less dormant than seeds from the other three trees when the variance due to the treatment X parent tree interaction caused by Tree 5 was removed ( P = 0.001 ). In contrast, there were no significant differences in germination response among parent trees in an ANOVA of just the two best pretreatments, meaning that although differences in dormancy existed between parent trees, both nicking and sulfuric acid pretreatments improved the germination response for all trees. Large differences in speed of germination occurred between treatments ( P = 0.001 ). The sulfuric acid and nicking pretreatments yielded the quickest response; 50% germination was reached in less than 5 days. Both the milling and water soak pretreatments were considerably slower and took 12-13 days to reach 50% germination. There was no significant difference due to parent tree in speed of germination. The tetrazolium test indicated that there were significant differences between treatments in the percentage of live seed ( P = 0.001 ). Single degree of freedom contrasts indicated that the 8 h water soak pretreatment had significantly lower percentage live seed (50%) compared with either the coffee grinder (74%) or control (83%) ( P = 0 . 0 0 2 ) . The high proportion of dead seed in the water pretreatment indicated that this treatment was too severe. Seeds from all pretreatments imbibed between 33 and 83% of their dry weight in water after 24 h compared with the control, which imbibed 12% (Fig. 4). Pretreatment significantly affected the rate of water uptake ( P = 0.001 ). Seeds pretreated with sulfuric acid, nicking or grinding absorbed a larger proportion of water than did seeds pretreated with a water soak, indicating that some seeds remained dormant in the latter pretreatment. Scanning electron micrographs illustrated the action of the pretreatments on the ultrastructure of the seed coat. The sulfuric acid pretreatment, when compared with the control, severely pitted the entire seed coat surface and cracked the normally raised button-shaped hilum (Figs. 5 (a) and 5 (b) ). In contrast, soaking in water left the hilar region and seed coat surface undisturbed, but enlarged the opening in the micropyle. Both the coffee grinder and nick pretreatments left deep tears in the seed coat (Fig. 5 (c) ). No differences in the ultrastructure of the seed coat among parent trees were observed in the control or any of the pretreatments. DISCUSSION
Nicking and immersion in concentrated sulfuric acid were consistently the best pretreatment methods for improving percentage germination in all ex-
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A.H. TODD-BOCKARIE AND M.L. DURYEA
METHODS TO IMPROVE GERMINATION OF DIALIUM GUINEENSE
269
Fig. 5. Scanning electron micrographs (a) showing the deep cracking of the seed coat surface and button-shaped hilar cap in the control; (b) illustrating the tiny fissures created on the seed coat surface, pitting of the hilar cap and opening of the micropyle for seed immersed in concentrated sulfuric acid for 8 min; (c) demonstrating the deep tear in the seed coat tissue, but intact hilar cap for seed nicked with a scalpel. (Magnification, × 150). periments. Successful pretreatment with concentrated sulfuric acid has also been reported for the related species Dialium maingayi Baker in Malaysia (Sasaki, 1980). In countries where a seed distribution network is well established, sulfuric acid pretreatment is routinely used to quickly pretreat large quantities o f seeds by trained personnel at well-equipped centralized nurseries (Willan, 1985 ). Recent work with Faidherbia albida (Del.) A. Chev. and Leucaena leucocephala (Lam.) De Wit. indicates that seeds from both of these species can be pretreated at a larger central nursery and stored for later distribution for periods of up to 1 year without losing viability ( D u g u m a et al., 1988). Further research is needed to determine whether the degree of dormancy in any way limits storage capacity for this species. For the limitedresource nursery, sulfuric acid would not be r e c o m m e n d e d due to the inherent cost, safety considerations and limited availability of the solvent. Nicking would be the most effective pretreatment for small village nurseries. Although
270
A.H. TODD-BOCKARIE AND M.L. DURYEA
this pretreatment is very labor intensive, a few hundred seeds can efficiently be pretreated in a matter of hours. Both heat and water soaks are commonly applied pretreatments to overcome physical dormancy in hard-seeded legume species (Rolston, 1978; Werker, 1980; Willan, 1985 ). We found that extended periods of exposure to either heat or water decreased percentage germination in D. guineense. Tetrazolium test results indicated that a large proportion of the seeds were killed by these pretreatments. There were significant differences in germination response among parent trees in the control and less successful pretreatments, which could be due to either environmental or genetic factors. Such differences in dormancy among parent trees could explain why some researchers (D. Hartley and E.K. Alieu, personal communication, 1989) consider pretreatments necessary for regeneration of this species while others do not (Okafor, 1982). A detailed comparison of the ultrastructure of the seed coat revealed no apparent differences between seeds from the five parent trees. Other avenues, such as differences in the biochemical composition of the seed coat, could explain the mechanism for dormancy in this species. For example, seed coat color differences resulting from the distribution and levels of quinones and pectins in the testa of three seed species within the genus Pisum were related to the degree of dormancy (Werker et al., 1979). Differential dormancy, in which individual seeds of the same species exhibit different degrees of dormancy, is an adaptive trait in seasonal tropical forests (Vazquez-Yanes and Orozco Segovia, 1984). Seed ecology research in Malaysia with the related species Dialium maingayi indicates that under natural forest conditions, spotty seed germination commences several months into the rainy season, well after the majority of other species (Ng, 1978 ). D. maingayi seeds took between 1 and 70 days to germinate. Several authors (Ng, 1978; Hall and Swaine, 1980; Garwood, 1982) have postulated that differential dormancy is a survival strategy which allows viable seed to remain on the forest floor for an extended period of time until more favorable environmental conditions exist (i.e. reduced competition), or to allow seeds to survive passage through the digestive tract of a vertebrate for dispersal. Although limited, our results indicate that seeds of Dialium guineense do not survive ingestion by animals. To fully understand the nature of differential dormancy for this species, further research is needed under natural conditions to determine which specific environmental factors trigger germination. CONCLUSION
Nicking and concentrated sulfuric acid pretreatments effectively improved the germination response for seeds from both dormant and non-dormant parent trees. Sulfuric acid pretreatments are recommended for well-equipped
METHODS TO IMPROVE GERMINATION OF DIALIUM GUINEENSE
271
centralized nurseries, whereas nicking is most appropriate for small village nurseries. More extensive experimental work in the laboratory and under natural conditions, using a larger sample of parent trees over multiple seed-years, is needed to determine to what degree dormancy in this species can be attributed to either environmental or genetic factors. ACKNOWLEDGEMENTS
We would like to thank the Department of Forestry and the Center for African Studies at the University of Florida for supporting this research. We would like to express our sincere appreciation to the staff of Tiwai Island Wildlife Sanctuary for collecting the seeds. For reviewing the manuscript and for their technical assistance on the project we wish to thank Drs. S.H. West, T. White and P.K. Nair. In particular, we would like to recognize the following individuals for their ready willingness to assist on the project: Philip Bockarie, Bonnie Hammer, Kris Irwin, Karen Kainer, Deborah McGrath and Carol McCormac Wild. In addition, the constant technical assistance of Jean Thomas at the USDA-ARS Agronomy Seed Laboratory was most appreciated.
REFERENCES Ayensu, E.S., 1978. Medicinal Plants of West Africa. Reference Publishers, Algonac, MI, 156 PP. Cline-Cole, A.K., 1987. The socio-ecology of firewood and charcoal on the Freetown Peninsula. Africa, 57: 457-497. Dalziel, J., 1948. The Useful Plants of West Tropical Africa: Being an Appendix to the Flora of West Tropical Africa. J. Hutchinson and J. Dalziel, Crown Agents, London, 612 pp. Donnelly, E.D., Watson, J.E. and McGuire, J.A., 1972. Inheritance of hard seed in Vicia. J, Hered., 63: 361-365. Duguma, B., Kang, B.T. and Okali, D.U., 1988. Factors affecting germination ofleucaena ( Leucaena leucocephala (Lam.) De Wit seed. Seed Sci. Technol., 16: 489-500. Falconer, J. and Koppell, C., 1990. The Major Significance o f ' M i n o r ' Forest Products: Thc Local Use and Value of Forests in the West African Humid Forest Zone. Community Forestry Note No. 6, FAO, Rome, 232 pp. Garwood, N., 1982. Seasonal rhythm of seed germination in a semi-deciduous tropical forest. In: E.G. Leigh, A.S. Rand and D.M. Windsor (Editors), The Ecology of a Tropical Forest: Seasonal Rhythms and Long-term Change. Smithsonian Institution Press, Washington, DC, pp. 173-185. Gotz, E., 1983. Timber Trees of the Gambia. Stiftung Walderhaltung in Afrika, Hamburg, 104 PP. Gupta, B.N., Saxema, S.K. and Durra, B.K., 1973. Germination, seedling behavior and phytomass of some Acacia in the nursery stage. Indian For., 99: 352-358. Hall, J.B. and Swaine, M.D., 1980. Seed stocks in Ghanian forest soils. Biotropica, 12: 256263. Irvine, F., 1952. West African Botany. Oxford University Press, London, 204 pp. Kamara, J., 1986. Firewood Energy in Sierra Leone: Production, Marketing and Household Use
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Patterns. Studien fur Integrierten Landlichen Entwicklung No. 9, Verlag Weltarchiv GmbH., Hamburg, 227 pp. Kamveti, D., 1982. Tree Planting in Africa South of the Sahara. Environmental Liaison Centre, Nairobi, 75 pp. Kariuki, E.M. and Powell, G.R., 1988. Pretreatment and germination of seeds of three leguminous tree species indigenous to Kenya. Seed Sci. Technol., 16: 477-487. Lee, Choon-soon and Rawlings, J.O., 1982. Design of experiments in growth chambers--uniformity trials in the North Carolina State University phytotron. Crop Sci., 22:551-558. Longman, K.A. and Jenik, J., 1987. Tropical Forest and its Environment. Longman Scientific, London, 347 pp. Moore, R.P., 1985. Tetrazolium Testing Manual. International Seed Testing Association, Zurich, 99 pp. Msanga, H.P. and Maghembe, J.A., 1986. Effect of hot water and chemical treatments on the germination ofAlbizia schimperana seed. For. Ecol. Manage., 17:137-146. Ng, F.S.P., 1978. Strategies of establishment in Malayan forest trees. In: P.B. Tomlinson and M.H. Zimmerman (Editors), Tropical Trees as Living Systems. Cambridge University Press, London, pp. 129-162. Nyerges, A.E., 1987. The development potential of the Guinea Savanna: social and ecological constraints in the West African "Middle Belt". In: P.D. Little, M.M. Horowitz and A.E. Nyerges (Editors), Lands at Risk in the Third World: Local Level Perspectives. Westview Press, Boulder, CO, pp. 316-336. Nyerges, A., 1989. Coppice swidden fallows in tropical deciduous forest: biological, technological and sociocultural determinants of secondary forest successions. Hum. Ecol., 17 (4): 37940O. Nyoko, G.C., 1980. Weed control and nitrogen restoration with legumes in upland rice culture in Sierra Leone. In: R. Rosswell (Editor), Nitrogen Cycling in West African Ecosystems. UNEP, Stockholm, 450 pp., pp. 261-267. Okafor, J.C., 1978. Development of forest tree crops for food supplied in Nigeria. For. Ecol. Manage., 1: 235-247. Okafor, J.C., 1980a. Edible woody plants in the rural economy of Nigeria. Int. Tree Crops J., 1: 131-141. Okafor, J.C., 1980b. Edible indigenous woody plants in the rural economy of the Nigerian forest zone. For. Ecol. Manage., 3: 45-55. Okafor, J.C., 1982. Promising trees for agroforestry in southern Nigeria. In: L. MacDonald (Editor), Agroforestry in the African Humid Tropics. United Nations University Press, Tokyo, pp. 113-117. Okafor, LC. and Fernandes, E.C.M., 1987. Compound farms of southeastern Nigeria: A predominant agroforestry homegarden system with crops and small livestock. Agrofor. Syst., 5: 153-168. Okigbo, B., 1976. Role of the legume in small holdings of the humid tropics of Africa. In: NIFTAL (Editor), Proc. Syrup. Exploiting the Legume-Rhizobium Symbiosis in Tropical Agriculture. University of Hawaii Press, Honolulu, pp. 97-117. Olvera, E. and West, S.H., 1985. Aspects of germination ofLeucaena. Trop. Agric., 62: 68-72. Rolston, M.P., 1978. Water impermeable seed dormancy. Bot. Rev,, 44: 365-396. Saha, P.K. and Takahashi, N., 198 l. Seed dormancy and water uptake in Crotalaria servicea Reitz. Ann. Bot., 47: 423-425. Sasaki, S., 1980. Storage and germination of some Malaysian legume seeds. Malays. For., 43: 27-32. Savill, P. and Fox, R., 1968. Trees of Sierra Leone. Government of Sierra Leone, Freetown, 316 pP.
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Shannon, J.G., Wilcox, J.R. and Probst, A.H., 1972. Estimated gains from selection for protein and yield in the F4 generation of six soybean populations. Crop Sci., 12: 824-826. Sheikh, M.I., 1980. Effect of different treatments to hasten tree seed germination. Pak. J. For.. Oct, pp. 176-180. Snedecor, G. and Cochran, W., 1967. Statistical Methods. Iowa Slate University Press, Ames, IA, 593 pp. Szolnoki, T.W., 1985. Food and Fruit Trees of the Gambia. Stiftung Walderhaltung in Afrika, Hamburg, 132 pp. UNESCO/UNEP/FAO, 1978. Tropical Forest Ecosystems: A State of Knowledge Report. Unesco, Paris, 683 pp. Vazques-Yanes, C. and Orozco Segovia, A., 1984. Ecophysiology of seed germination in the tropical humid forests of the world: a review. In: E. Medina, H,A. Mooney and C. VazquesYanes (Editors), Physiological Ecology of Plants in the Wet Tropics. Junk, The Hague. pp. 37-50. Von Carlowitz, P., 1986. Multipurpose Tree and Shrub Seed Directory. ICRAF, Nairobi. 265 pp. Von Maydell, H., 1983. Trees of the West African Sahel. GTZ, Eschborn, 525 pp. Werker, E., 1980. Seed dormancy as explained by the anatomy of embryo envelopes. Isr. J. Bot., 29: 22-44. Werker, E., Marbach, I. and Mayer, A.M., 1979. Relation betweex~ the anatomy of the testa. water permeability and the presence of phenolics in the genus Pisup~7. Ann. Bot.. 43: 765771. West, S.H. and Marousky, F., 1989. Mechanism of dormancy in Pensacola Bahiagrass. Crop Sci., 29: 787-791. Willan, R., 1985. A Guide to Forest Seed Handling. FAO Forestry Paper 20/2, Rome, 379 pp.
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Seed pretreatment methods to improve germination of the multipurpose West African forest species Dialium guineense A.H. Todd-Bockarie and M.L. Duryea University of Florida, Department QfForestry, Gainesville, FL 3261 I, USA (Accepted 7 May 1992 )
ABSTRACT Todd-Bockarie, A.H. and Duryea, M.L., 1993. Seed pretreatment methods to improve germination of the multipurpose West African forest species Dialium guineense. For. Ecol. Manage., 57: 257273. Two experiments preceded by a pilot study were conducted to determine the optimal method for overcoming seed coat dormancy of Dialium guineense Willd. Seeds from parent trees were kept separate to determine whether genetic variation in dormancy influenced pretreatment choice. There were pronounced differences between parent trees in germination response for the control and the two lower-germinating pretreatments, indicating that differential dormancy exits within this species. Nicking and concentrated sulfuric acid pretreatments effectively improved the germination response for seeds from both dormant and non-dormant parent trees. Sulfuric acid pretreatments are recommended for well-equipped centralized nurseries, whereas nicking is most appropriate for small village nurseries.
INTRODUCTION
Dialium guineense Willd., or velvet tamarind, is a multipurpose tree of the West African Guinean Forest zone. It typically grows in dense savanna and riparian forests, ranging from Senegal to the Sudan, along the southern boundary of the Sahel (Szolnoki, 1985). It is a leguminous species and belongs to the Caesalpiniaceae family. The velvety-black fruit of Dialium guineense contains from one to five hard seeds surrounded by a bright orangered pulp. Fruits ripen in April to May during the dry season (Savill and Fox, 1968 ). Elephants, birds, monkeys and humans disperse the seeds when they eat the acid-tasting astringent fruit pulp (Dalziel, 1948; Irvine, 1952 ). At least three primate species (Spot-nosed monkey (Cercopithecus nictitans), CampCorrespondence to."A.H. Todd-Bockarie, University of Florida, Department of Foresty, Gainesville, FL 32611, USA.
© 1993 Elsevier Science Publishers B.V. All rights reserved 0378-1127/93/$06.00
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bell's monkey (Cercopithecus campbelli) and the Chimpanzee (Pan troglodytes) ) frequently feed on Dialium fruit (M. Conteh, personal communication, 1989).
Dialium guineense serves two important functions for the West African farming family. First, it is an energy-efficient and preferred charcoal species for rural villagers who depend on wood for all their energy needs (Kamara, 1986; Cline-Cole, 1987 ). Second, the fruits and leaves are consumed in critical food shortage periods during the dry season when farm produce is unavailable (Okafor, 1978; Falconer and Koppell, 1990). In addition to these two primary functions, the tree provides a variety of different products throughout the cropping calendar (Table 1 ). Given the pressing need for increased food production in many West African nations, improvements in tree crop production methods for this species are needed. Under minimal tillage swidden agricultural operations, the tree regenerates naturally during the fallow period (Nyerges, 1989). Currently, farmers wait for volunteer seedlings to produce harvestable fruits in farm fallows (Okafor, 1980b). However, as pressure on available land resources inTABLE 1 Multiple uses of Dialium guineense by West African farm families Part
Description of use
Reference
Fruit
Edible fruits rich in Vitamin C A thirst-quenching beverage Processed into jams and jellies Income supplement for women
Okafor, 1978 Okafor and Fernandes, 1987 Okafor, 1980a Kamara, 1986
Leaves
Vegetable (fermented for flavor) Fodder for livestock Medicine for diarrhea Mulch Eye ointment
Szolnoki, 1985 Okigbo, 1976 Ayensu, 1978 Von Carlowitz, 1986 Dalziel, 1948
Wood
Charcoal Fuelwood Resistant to termites Poles and bridge construction Carvings, utensils and tools
Cline-Cole, 1987 Kamara, 1986 Gotz, 1983 Gotz, 1983 Okigbo, 1976
Bark
Mouthwash and toothache cure
Ayensu, 1978
Branches
Toothbrushes or chewsticks Stakes for trailing yams Hedgerow and windbreak
Dalziel, 1948 Okigbo, 1976 Von Carlowitz, 1986
Roots
Erosion control
Nyoko, 1980
METHODS TO IMPROVE GERMINATION OF DL4LIUM GUINEENSE
259
creases and fallow periods shorten, secondary succession for tree species is inhibited and grasses and forbs are favored (Unesco/UNEP/FAO, 1978; Nyerges, 1987; Longman and Jenik, 1987 ). The lack of information on appropriate regeneration methods limits the use of this species in reforestation activities. Regeneration of Dialium from seed is difficult because seed coat dormancy inhibits germination. Poor germination may be linked to the rupturing of the distinctive waxy cap in the hilar region, which is directly connected to the fiat, disk-shaped embryo in the interior of the seed. Seed pretreatment is needed for quick, uniform germination. A range of different seed pretreatment methods have been successfully used in rupturing the water-impermeable testa of other tree species to overcome dormancy (Rolston, 1978; Werker, 1980). Chemical pretreatments such as immersion for several minutes in concentrated sulfuric acid have been particularly effective in overcoming physical dormancy in other leguminous species (Von Carlowitz, 1986). However, laboratory grade acids are expensive, difficult to obtain and pose a safety problem when used in village nurseries (Willan, 1985). In West Africa there are several alternative pretreatment methods which may be both effective in breaking seed coat dormancy and widely available in the rural villages. Strong acids found in the digestive tract of domestic livestock are one possibility. Ruminants have been employed as a pretreatment for hard seed with varying levels of success in several countries including Senegal for Adansonia digitata L. (Von Maydell, 1983 ), Kenya for Acacia tortilis (Forsskal) Hayne. (Kamveti, 1982), and Pakistan for Cassia fistula L. (Sheikh, 1980). By varying immersion time and temperature, hot water treatments have effectively been used to break seed coat dormancy in several related legumes (Werker, 1980). Mechanical pretreatments such as nicking, sanding, aging, threshing and burning have also improved percentage germination in many hard-seeded species (Msanga and Maghembe, 1986; Kariuki and Powell, 1988; Duguma et al., 1988; West and Marousky, 1989). Genetic variation in seed characteristics is commonly exploited by plant breeders to create high-yielding cultivars for very specific purposes. For example, geneticists have effectively increased both the protein and the oil content of soybean seeds to produce a more nutritional product for the market (Shannon et al., 1972 ). Similarly, a genetic basis for seed coat impermeability evident in many agronomic species allows plant breeders to reduce the proportion of hard seed in a hybrid to promote quick, uniform germination (Rolston, 1978). The inter-relationship between environmental and genetic factors in controlling variation in seed coat permeability is not fully understood. However, several authors have noted distinct differences among seed sources of hard-seeded leguminous tree species to pretreatments for overcoming dormancy which may imply some genetic basis for impermeability ( Don-
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A.H. TODD-BOCKARIE AND M.L. DURYEA
nelly et al., 1972; Gupta et al., 1973; Olvera and West, 1985 ). Given that over 85% of legume species possess the hardseededness trait (Rolston, 1978 ), manipulation of seed coat permeability by breeders would have a significant impact on nursery practices by decreasing or eliminating the need for pretreatments. The objectives of the present study were two-fold. First, a wide range of pretreatment methods appropriate for village nurseries in West Africa were tested to establish the optimum germination response. Second, we sought to determine whether genetic variability in seed coat dormancy existed for the species, and ascertain what effect this may have on pretreatment choice. METHODS
Seed collection Seed was collected from 10 open-pollinated Dialium guineense trees at the close of the dry season in March 1989 by Peace Corps Volunteers, researchers and Tiwai Island Wildlife Sanctuary staff. The collection area was located in Barri and Koya Chiefdoms, Sierra Leone (45 km inland from the Atlantic Ocean in the southeastern forest region; latitude 7°N, longitude 7 °W). A minimum distance of I km was maintained between trees so that each collection would represent a separate genetic line. Fruits were cleaned and seeds with obvious insect and fungal infestations were removed. After a 7-day drying period, seeds were packaged separately by parent tree and shipped by air to the University of Florida, Gainesville (USDA Permit 37-72360). All seed was stored in sealed silica-gel-lined containers in an air-conditioned room at 18 ° C until being used in the experiments.
Germination All germination tests were conducted using a Percival 1-30B plant growth chamber under a controlled fluorescent light and a temperature regime of 25 ° C, with a 12 h day/night cycle, to approximate growing conditions in Sierra Leone. A series of pilot studies and two experiments were conducted to test 39 pretreatments (Table 2). A bulk seedlot, which was a mixture of seeds from five different parent trees (Nos. 6-10), was used in Experiment 1. A second group of five different parent trees (Nos. 1-5 ) was tested separately in Experiment 2. The most promising methods were retained from the pilot studies for the experiments. We blocked by shelf in the germination chamber to minimize differences due to gradients in humidity and temperature (Lee and Rawlings, 1982 ). Seeds from each parent tree were surface-sterilized for 5 rain in a 20% chlorine solution prior to treatment. Treated seeds were sown in 9 mm petri plates lined with 2 layers of filter paper. Each plate was sealed
METHODS TO IMPROVE GERMINATION OF DIALIUM GUINEENSE
261
TABLE 2 Thirty-nine seed pretreatment m e t h o d s employed during the pilot study and Experiments 1 and 2 Number'
Pretreatment description
Pilot
Experiment
1
Control
x
x
Chemical 2, 3, 4 5, 6, 7, 8 9, 10, 11
95% ethanol: 1, 3 or 6 h H2SO4: 1, 7, 8 or 10 min H2SO4: 2, 4 or 6 min
x
Heal 12 13,14,15 16,17,18 19,20,21 22 23,24,25 26 27,28,29,30
Warm H20:5 min at 80°C Warm H20: 2, 4 or 6 min at 80°C Boiling H20: l, 2 or 3 min at 100 ° C Shock2: l, 2 or 3 min at 1 0 0 ° C / 3 4 ° C Cold H20 soak: 48 h at 25 °C Hot H20 soak: 6, 8 or 24 h at 100°C Dry heat: 15 min at 100°C Fire: 0.5, 1, 1.5 or 7 min at 2400°C
x
x x
x x x x x x x x
Meehanical 31 32, 33 34, 35
Nicking Commercial mill: 1 or 2 min Coffee grinder: 1 o r 2 s
x x
Combination 36 37, 38, 39
Sheep ingestion Accelerated aging3: 2, 4 or 6 days
x x
x
x
x x
~Consecutive numbers on a line represent several pretreatment combinations. 2Shock was applied by immersing the seeds in boiling water followed by ice water for the specified time period. 3Accelerated aging consisted o f placing seeds in a controlled environment o f 100% relative humidity over a water bath maintained at 40 °C for several days.
with Parafilm after 10 ml of distilled water had been added. Germination, defined as the emergence of the radicle beyond 2 mm, was recorded daily for a 14-day period.
Tetrazolium chloride test procedure At the close of each germination experiment, the viability o f all ungerminated seeds were assessed using a tetrazolium chloride test. The testa of each seed was nicked with a scalpel before being allowed to fully imbibe distilled water over a 24 h period at 20 ° C following the methodology of Moore ( 1985 ). The seed coat was then removed from the imbibed seed and a longitudinal
262
A.H. TODD-BOCKARIEAND M.L. DURYEA
TABLE 3 Topographical stain evaluation classes (Moore, 1985 ) Class
Description
Viability
1 2 3 4 5
Uniform red staining of embryo and radicle Pale pink staining of embryo and radicle Cotyledons < 50% unstained Radicle unstained or damaged No staining
Germinable Germinable Weak germination Non-germinable Non-germinable
section through the endosperm was cut to allow the embryo tissue to imbibe the stain. Prepared seeds were immersed in a 1% solution of 2,3,5-triphenyl tetrazolium chloride for 9 h in the dark at 35 °C. The topographical staining pattern of the embryo, cotyledons, radicle and endosperm was evaluated for each seed and used to place the seed into one of five viability classes (Moore, 1985 ) (Table 3 ). Percentage live seed for each treatment X parent tree combination was calculated by first adding together the number of seeds in the three germinable evaluation classes ( 1-3 ) and then dividing this sum by the total number of seeds.
Pilot studies A series of non-replicated pilot studies were conducted on a bulk seed lot to initially test and eliminate pretreatment methods which did not alter seed coat dormancy. Pretreatment methods were grouped into four categories as follows: (1)chemical; ( 2 ) h e a t ; (3)mechanical; (4)combinations which consisted of two or more types of treatments to the seed coat (Table 2 ). Separate 50-seed samples were used in testing a range of treatment combinations within each category. A total of 14 pretreatment combinations were screened in order to find the most promising treatment combinations in improving percentage germination.
Experiment I Using a bulk seedlot which was a mixture of seeds from five parent trees (Nos. 6-10), 25 pretreatment combinations were tested in Experiment 1 (Table 2). The most promising treatments from the previous pilot studies were used. A randomized complete block design was employed with 25 pretreatments blocked by shelf in the growth chamber (1500 total seeds= 4 blocks X 25 pretreatments X 15 seeds).
METHODS TO IMPROVE GERMINATION OF DIA LIUM G UINEENSE
263
Experiment 2 The four pretreatment combinations with the best percentage germination together with a control were selected for testing with parent trees (Nos. 1-5 ) in Experiment 2 (Table 2). A randomized complete block design was employed with four blocks, five parent trees and four pretreatments: ( 1 ) nicking; (2) 8 min sulfuric acid immersion; (3) 8 h soaking in hot water; (4) 1 s grating in a coffee grinder (1500 total seeds = 4 blocks × 5 pretreatments X 5 parent trees × 15 seeds). Imbibition measurements and scanning electron microscopy were utilized to characterize parent tree X pretreatment interactions.
Imbibition measurements Water uptake was measured for two seeds sampled randomly from each of the five parent trees and five treatments in all four blocks in Experiment 2. Samples were taken during the imbibition (Phase A) and activation (Phase B) stages of germination. The five time intervals for sampling were ( 1 ) control prior to treatment, and (2) 0 h, (3) 8 h, (4) 16 h and (5) 24 h after treatment. The fresh weight of each seed sample was measured using a Sartorius H-51 analytical balance. After being dried for 48 h at 70°C, each sample was then reweighed. The percent water absorption was calculated for all samples based on the initial moisture content of the seed following the method of Saha and Takahashi ( 1981 ).
Scanning electron microscopy A sample of four seeds was randomly selected from every parent tree in each of the five treatments in Experiment 2 and dried in a vacuum-sealed desiccator before mounting on a l u m i n u m stubs. Each seed was sputter coated with gold for 6 min before being viewed in a Hitachi 450-S scanning electron microscope at an accelerating potential of 20 kV. Disruption of the strophiole, hilum, micropyle and testa were recorded for each seed. Representative seed samples were photographed for each treatment.
Statistical analys& An analysis of variance (ANOVA) was conducted on percentage germination for all germination tests. The data were transformed and arcsin values were used in performing the analysis (Snedecor and Cochran, 1967). Duncan's multiple range test was used to test the significance of general treatment and parent tree effects, and single degree of freedom contrasts were used to determine the significance between specific treatment and parent tree comparisons. All treatments were considered statistically significant at P = 0.01.
264
A.H. TODD-BOCKARIE AND M.L. DURYEA
Treatment and parent tree differences in water absorption rates for the imbibition measurements were analyzed using an ANOVA at each time interval. An additional ANOVA was performed on water absorption combined across all five time periods because the mean square error (MSE) for all intervals were comparable; time period was treated as a sub-plot factor in a split-plot design. RESULTS
Pilot studies Nicking the seed coat surface with a scalpel produced the highest percentage germination (96%) compared with the control (10%). The three most promising treatments were the coffee grinder, sulfuric acid and water soak pretreatments. Substantially higher percentage germination resulted from 2 s of grinding (65%) compared with only 1 s (10%). Immersion for 7 min in concentrated sulfuric acid (55%) was the next most promising pretreatment, with the 1 min immersion (25%) and the 10 min immersion (4%) producing considerably lower germination values. Temperature appeared to affect the response to water soak pretreatments. A quick 5 min immersion in water at 80 °C (32%) was more effective than a longer cold water soak at 25 °C (8%). Extreme heat pretreatments, such as 7 min fire and 15 min dry heat ( 100 ° C), killed the seeds. Employing the digestive tract of sheep as a pretreatment was equally ineffective; all the seeds were digested.
Experiment 1 Five of the 25 pretreatments produced encouraging germination responses, including nicking, sulfuric acid immersion and water soak (Fig. 1 ). The nicking and sulfuric acid pretreatments were significantly better than the remaining treatments (P=0.001) or the control (P=0.001). Nicking produced the best germination response overall, and was significantly higher than any of the sulfuric acid treatments ( P = 0.001 ). The 6 h water soak resulted in 22% germination, which was not statistically different from the control (10%). However, it was viewed as promising if further refined. Twenty of the 25 pretreatments resulted in germination responses equal to, or lower than, the control. The tetrazolium test indicated that the extreme heat treatments killed a significant proportion of the seed ( P = 0.001 ) (Fig. 2 ). As illustrated by the fire, boiling water and shock pretreatments, the longer the exposure time to heat, the greater the proportion of dead seed. In contrast, an equal proportion of the seed remained alive when compared with the control for the aging, ethanol soak, and warm water treatments, although the combinations used did not overcome dormancy.
METHODS TO IMPROVEGERMINATIONOF DIALIUM GUINEENSE
265
100 95
90 85
a
80 75 70
._o 65 60 55 50 45
u
b
b
40 35 30
25
¢
0
Seed Pretreatment
Fig. 1. Mean percentage germination for the twelve most promising seed pretreatment methods compared with the control for Experiment 1. Pretreatments with the same letter were not significantly different. 100 90 95% LSD I 80
7O 60 "0 a) 50
Or) Q
_> 40 ,-I
30 20 10 0
.o j . ~ o ~ . ~ o ~ - o
,, ~o-,~ ~ , , ~ , , ~ : ~ , , ~ ,
~ ~. ~ ~
Treatment
Fig. 2. Mean percentages o f live seed by p r e t r e a t m e n t c o m p a r e d with the control for the tetrazolium chloride test in Experiment I.
266
A.H. TODD-BOCKARIE
90 10070
A N D M.L. D U R Y E A
Tree 1
o~60
Tree 2
C O
Tree 3
~60
U,
Q
Tree 4
I Tree 5
.... • ....
• 5o
I--
30
b"
e= 20 n 10 0O
I 20 3040
li0
~)
60
70
60
9~ ,100
CONTROL 1120 GRIND
Pretreatment
NICK 112SO4
Germination
(%)
Fig. 3. Differences in mean percentage germination among five parent trees for four pretreatment methods compared with the control in Experiment 2.
100 95% LSD = CONTROL "
90
I
NICK --"@o-
/
80
GRIND ....G....
//
HZS04
70
!
I
//
/
/
A V
H20 SOAK
60
--A.-.
/
~
/
//
/
IN/
~. so
/
/
E
/ /
,
/
/
/
t
i
/:' /:" t-Y ~:.
,&. . . . . . . . . . . . . .
•
30
/
/
/
20
/ /
/
/ / /
k"" ..--
.oO°°
10
HR
I
8 HR Time
I
16 HR
I 24 HR
(Hours)
Fig. 4. A comparison among water uptake patterns for the control and four pretreatment methods during the first 24 h of imbibition in Experiment 2.
Experiment 2 A satisfactory mean germination response was obtained for the nicking (90%) and sulfuric acid (93%) pretreatments compared with the control
METHODS TO IMPROVE GERMINATION OF DIALIUM GUINEENSE
267
(29%), 1 s grating in a coffee grinder (50%), or 8 h water soak (36%). There were pronounced differences between parent trees in germination response for the control and the two lowest-germinating pretreatments (Fig. 3 ). A separate ANOVA was used to compare the germination responses of different parent trees within the control and the two lowest-germinating pretreatments. Seeds from parent trees Nos. 4 and 5 were significantly less dormant than seeds from the other three trees when the variance due to the treatment X parent tree interaction caused by Tree 5 was removed ( P = 0.001 ). In contrast, there were no significant differences in germination response among parent trees in an ANOVA of just the two best pretreatments, meaning that although differences in dormancy existed between parent trees, both nicking and sulfuric acid pretreatments improved the germination response for all trees. Large differences in speed of germination occurred between treatments ( P = 0.001 ). The sulfuric acid and nicking pretreatments yielded the quickest response; 50% germination was reached in less than 5 days. Both the milling and water soak pretreatments were considerably slower and took 12-13 days to reach 50% germination. There was no significant difference due to parent tree in speed of germination. The tetrazolium test indicated that there were significant differences between treatments in the percentage of live seed ( P = 0.001 ). Single degree of freedom contrasts indicated that the 8 h water soak pretreatment had significantly lower percentage live seed (50%) compared with either the coffee grinder (74%) or control (83%) ( P = 0 . 0 0 2 ) . The high proportion of dead seed in the water pretreatment indicated that this treatment was too severe. Seeds from all pretreatments imbibed between 33 and 83% of their dry weight in water after 24 h compared with the control, which imbibed 12% (Fig. 4). Pretreatment significantly affected the rate of water uptake ( P = 0.001 ). Seeds pretreated with sulfuric acid, nicking or grinding absorbed a larger proportion of water than did seeds pretreated with a water soak, indicating that some seeds remained dormant in the latter pretreatment. Scanning electron micrographs illustrated the action of the pretreatments on the ultrastructure of the seed coat. The sulfuric acid pretreatment, when compared with the control, severely pitted the entire seed coat surface and cracked the normally raised button-shaped hilum (Figs. 5 (a) and 5 (b) ). In contrast, soaking in water left the hilar region and seed coat surface undisturbed, but enlarged the opening in the micropyle. Both the coffee grinder and nick pretreatments left deep tears in the seed coat (Fig. 5 (c) ). No differences in the ultrastructure of the seed coat among parent trees were observed in the control or any of the pretreatments. DISCUSSION
Nicking and immersion in concentrated sulfuric acid were consistently the best pretreatment methods for improving percentage germination in all ex-
268
A.H. TODD-BOCKARIE AND M.L. DURYEA
METHODS TO IMPROVE GERMINATION OF DIALIUM GUINEENSE
269
Fig. 5. Scanning electron micrographs (a) showing the deep cracking of the seed coat surface and button-shaped hilar cap in the control; (b) illustrating the tiny fissures created on the seed coat surface, pitting of the hilar cap and opening of the micropyle for seed immersed in concentrated sulfuric acid for 8 min; (c) demonstrating the deep tear in the seed coat tissue, but intact hilar cap for seed nicked with a scalpel. (Magnification, × 150). periments. Successful pretreatment with concentrated sulfuric acid has also been reported for the related species Dialium maingayi Baker in Malaysia (Sasaki, 1980). In countries where a seed distribution network is well established, sulfuric acid pretreatment is routinely used to quickly pretreat large quantities o f seeds by trained personnel at well-equipped centralized nurseries (Willan, 1985 ). Recent work with Faidherbia albida (Del.) A. Chev. and Leucaena leucocephala (Lam.) De Wit. indicates that seeds from both of these species can be pretreated at a larger central nursery and stored for later distribution for periods of up to 1 year without losing viability ( D u g u m a et al., 1988). Further research is needed to determine whether the degree of dormancy in any way limits storage capacity for this species. For the limitedresource nursery, sulfuric acid would not be r e c o m m e n d e d due to the inherent cost, safety considerations and limited availability of the solvent. Nicking would be the most effective pretreatment for small village nurseries. Although
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this pretreatment is very labor intensive, a few hundred seeds can efficiently be pretreated in a matter of hours. Both heat and water soaks are commonly applied pretreatments to overcome physical dormancy in hard-seeded legume species (Rolston, 1978; Werker, 1980; Willan, 1985 ). We found that extended periods of exposure to either heat or water decreased percentage germination in D. guineense. Tetrazolium test results indicated that a large proportion of the seeds were killed by these pretreatments. There were significant differences in germination response among parent trees in the control and less successful pretreatments, which could be due to either environmental or genetic factors. Such differences in dormancy among parent trees could explain why some researchers (D. Hartley and E.K. Alieu, personal communication, 1989) consider pretreatments necessary for regeneration of this species while others do not (Okafor, 1982). A detailed comparison of the ultrastructure of the seed coat revealed no apparent differences between seeds from the five parent trees. Other avenues, such as differences in the biochemical composition of the seed coat, could explain the mechanism for dormancy in this species. For example, seed coat color differences resulting from the distribution and levels of quinones and pectins in the testa of three seed species within the genus Pisum were related to the degree of dormancy (Werker et al., 1979). Differential dormancy, in which individual seeds of the same species exhibit different degrees of dormancy, is an adaptive trait in seasonal tropical forests (Vazquez-Yanes and Orozco Segovia, 1984). Seed ecology research in Malaysia with the related species Dialium maingayi indicates that under natural forest conditions, spotty seed germination commences several months into the rainy season, well after the majority of other species (Ng, 1978 ). D. maingayi seeds took between 1 and 70 days to germinate. Several authors (Ng, 1978; Hall and Swaine, 1980; Garwood, 1982) have postulated that differential dormancy is a survival strategy which allows viable seed to remain on the forest floor for an extended period of time until more favorable environmental conditions exist (i.e. reduced competition), or to allow seeds to survive passage through the digestive tract of a vertebrate for dispersal. Although limited, our results indicate that seeds of Dialium guineense do not survive ingestion by animals. To fully understand the nature of differential dormancy for this species, further research is needed under natural conditions to determine which specific environmental factors trigger germination. CONCLUSION
Nicking and concentrated sulfuric acid pretreatments effectively improved the germination response for seeds from both dormant and non-dormant parent trees. Sulfuric acid pretreatments are recommended for well-equipped
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centralized nurseries, whereas nicking is most appropriate for small village nurseries. More extensive experimental work in the laboratory and under natural conditions, using a larger sample of parent trees over multiple seed-years, is needed to determine to what degree dormancy in this species can be attributed to either environmental or genetic factors. ACKNOWLEDGEMENTS
We would like to thank the Department of Forestry and the Center for African Studies at the University of Florida for supporting this research. We would like to express our sincere appreciation to the staff of Tiwai Island Wildlife Sanctuary for collecting the seeds. For reviewing the manuscript and for their technical assistance on the project we wish to thank Drs. S.H. West, T. White and P.K. Nair. In particular, we would like to recognize the following individuals for their ready willingness to assist on the project: Philip Bockarie, Bonnie Hammer, Kris Irwin, Karen Kainer, Deborah McGrath and Carol McCormac Wild. In addition, the constant technical assistance of Jean Thomas at the USDA-ARS Agronomy Seed Laboratory was most appreciated.
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