Mutete, Mazarura, Gasura, Richardson-Kageler, Ngadze and Kativu (2019). Seed Science and Technology, 47, 3, 377-383. https://doi.org/10.15258/sst.2019.47.3.13
Research Note
Hyphaene petersiana dormancy and germination
P. Mutete1, U. Mazarura2*, E. Gasura2, S. Richardson-Kageler2, E. Ngadze2 and S. Kativu3
1 Forest Research Centre, 1 Orange Grove Drive, Box HG 595, Highlands, Harare, Zimbabwe 2 Department of Crop Science, University of Zimbabwe, Harare, Zimbabwe 3 Department of Biological Sciences, University of Zimbabwe, Harare, Zimbabwe * Author for correspondence (E-mail: [email protected])
(Submitted November 2018; Accepted August 2019; Published online December 2019)
Abstract
Hyphaene petersiana is a naturally occurring palm tree in Zimbabwe. The tree provides an important supplementary source of income to the communities where it is found, with a high demand for foliage for making baskets and sap for wine making. Propagation is both by vegetative and sexual methods, however, sexual propagation is constrained by the inherent dormancy of the seeds. Breaking seed dormancy is also the first step towards developing a conservation programme for the tree. The effect of water soaking (for 12, 24 and 48 hours), chemical scarification (sulphuric acid for 10 minutes), de-husking (tegument removal), sand papering as well as chemical treatment (thiourea and potassium nitrate) were compared with untreated seeds (control). No germination was observed for seeds treated with sulphuric acid. Water soaking for 12 hours gave the highest germination (71.7%) although not significantly different to other treatments.
Keywords: germination, Hyphaene petersiana, morphophysiological dormancy, synchronisation index
Experimental and discussion
H yphaene petersiana Mart ex Klortsch. is a wild palm tree commonly known as Ilala (Foote et al., 2003) that is native to the tropical regions of the African continent (Aguir and Aguiar, 1998). It grows to an average height of 18 m with characteristic fan-shaped leaves which can extend to 1.5-2 m long, (Cunningham and Milton, 1987). The tree is widely used for wine tapping (Sullivan et al., 1995; Sola et al., 2006), and its leaves serve as an important raw material for various craft objects (Doren, 1997; McKean, 2003), for construction purposes or as fodder for animals (Konstant et al., 1995). It is a dioecious
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377 P. MUTETE, U. MAZARURA, E. GASURA, S. RICHARDSON-KAGELER, E. NGADZE AND S. KATIVU plant with a male to female ratio of 1:2 (Sullivan et al., 1995; Sola et al., 2006) and the female can produce an average of 450 fruits per season (Fanshawe, 1967). Propagation is both by vegetative and sexual methods, however, sexual propagation is constrained by the inherent dormancy of the seeds (Orozco-Segovia et al., 2003). In the wild, passage of the seeds through the gut of wild animals helps to weaken the seed coat, thus removing dormancy and promoting germination (Fanshawe, 1967; Orozco-Segovia et al., 2003). Artificial methods include scarification and the use of chemicals such as thiourea (Newton, 2007), potassium nitrate or hormones such as gibberellic acid (GA3), to overcome physiological dormancy (Finch-Savage and Leubner-Metzger, 2006; Fransen et al., 2015). Thiourea has been used extensively for dormancy breaking (Amen, 1968; Kepczynski and Kepczynska, 1997; Newton, 2007) and works by stimulating biological activity in the embryo (Poljakoff and Mayer, 1960). Specifically, Gul (1998) noted that thiourea stimulates biological activity by countering the effect of ABA and lowering the levels of cytokinins in plant tissues. In spite of its socio-economic importance, few attempts have been made to artificially regenerate Hyphaene petersiana trees either from seeds or through vegetative means. The tree has survived in the wild but is threatened by population increase leading to agricultural land expansion. This is exacerbated by frequent droughts due to climate change. This puts pressure on the remaining tree population mainly due to over-exploitation. Therefore, there is need to improve its conservation, both in-situ and ex-situ. The seeds of H. petersiana exhibit dormancy (Sullivan et al., 1995), and according to Baskin and Baskin (2013), dormancy is a common feature in the Aceraceae family, which may affect ex-situ conservation. In the current study, the effect of different seed pre-treatment methods on their potential to release the dormancy of H. petersiana seeds were evaluated. For this study, mature seeds were collected from the south east lowveld of Zimbabwe between −20.418° N and −22.444° S, and 32.447° E and 30.951° W, during a field survey conducted in 2016. The seed lot consisted of a combination of seeds picked from the ground as well as seeds from mature fruits knocked down from the tree. For seeds picked from the ground, care was taken to collect only seeds for the current season which were easily distinguished by their fresh, shiny and intact exocarp. Soon after collection, the seeds were kept in a cold room at 25 ± 2°C until the experiments commenced. Before experiments commenced, parenchyma cells were removed by immersing the fruits in water overnight to ferment the cells before scraping them off with a knife. The seeds were then thoroughly mixed and divided into nine equal subsamples of 60 seeds each. Each subsample was then subjected to a unique pre-treatment method. Three seed lots were pre-treated separately by soaking in water for 12, 24 and 48 hours. Another sample was de-husked by cutting the shell using a hacksaw blade without injuring the embryo. Another sample was treated with concentrated sulphuric acid (95%) for 10 minutes (Meerow and Broschat, 2015) with continuous stirring. This was followed by rinsing under running tap water for 20 minutes. The other two samples were submerged either in thiourea or potassium nitrate for one hour. The final sample was spot sand- papered on the surface until the kernel was exposed. Untreated seeds were used as the control.
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Each treatment was further subdivided into four parts of 15 seeds each. These were then moistened and put in a clear empty plastic paper bag and then sealed after adding
15 ml of de-ionised water. The de-ionised water provided the moisture required for germination during the entire period of the experiment. The experiment was then conducted in a completely randomised design in an incubator. The incubator was kept at
25 ± 3°C and 70-75% relative humidity under continuous light. Germination was recorded when the tip of the radicle had grown free from the seed coat (Fariman et al., 2011; Neto et al., 2014). Weekly germination counts were made but terminated when germination was not observed for three weeks. Measured traits included germination percentage, mean germination time and synchronisation index, calculated using the GerminaQuant software 1.0 (Marques et al., 2015). Mean germination time measures mean length of incubation time and was calculated according to Ranal and Santana (2006) as follows:
k niti ∑ = 1 t = i k ni ∑ i = 1
Where, t is the mean germination time; ti the time from the start of the experiment to the th th i observation (day); ni number of germinated seeds in the i time; and k is the last time of observation. Germination synchrony is an adaptation of the Shannon index and measures the uncertainty associated with the distribution of relative frequency of germination (Ranal and Santana, 2006). Low values indicate a more synchronised germination process and is measured in bits representing a binary measure which counts germinated/non-germinated seeds. k n f log f , i , U = − ∑ i 2 i fi = k ∑ n i = 1 i i = 1 where fi is the relative frequency of germination; ni is the number of seeds germinated in the ith time; and k is the last day of germination. The data were subjected to ANOVA using GerminaR package for the statistical software application R and means were compared using the Least Significance Difference method (LSD) at 95% probability. None of the seeds pre-treated with concentrated sulphuric acid germinated over the entire incubation period and were therefore removed from the analysis. Significant differences were observed for mean number of seeds that germinated as well as for germination percentage (P < 0.05) amongst the treatment methods used (table 1). Germination varied between 10 and 71.7% with high values observed for seeds pre-treated by soaking in water for 12 and 24 hours as well as those that were treated with thiourea and potassium nitrate. Amongst the pre-treatment methods, the highest germination was recorded for seeds soaked in water for 12 hours (71.7%). Seeds that were de-husked had the lowest germination (table 1). De-husking and sand papering were associated with germination uncertainty equal to zero although they were not significantly different from the seeds treated by soaking in water for 12 and 24 hours as well as for both chemical methods.
379 P. MUTETE, U. MAZARURA, E. GASURA, S. RICHARDSON-KAGELER, E. NGADZE AND S. KATIVU
Table 1. Effects of pre-treatment methods on germination percentage, mean germination time (MGT) and synchronisation index (U) of Hyphaene petersiana seeds.
Germination Mean Germination Time Synchronisation Index Treatment (%) (weeks) (bite)
Control 51.7 ab 4.75 ab 0.41 ab De-husked 10.0 c 8.00 a 1.00 a
a ab ab H2O soak 12 hours 71.7 4.59 0.43
a ab ab H2O soak 24 hours 70.0 5.00 0.57
ab b b H2O soak 48 hours 48.3 3.45 0.25 95% sulphuric acid 10 minutes 0 – – Potassium nitrate 1 hour 61.7 a 4.65 ab 0.35 ab Sand papered 26.7 bc 5.50 ab 1.00 a Thiourea 1 hour 66.7 a 5.50 ab 0.41 ab Treatments with the same letter are not signifi cantly different using 5% LSD (P < 0.05).
Untreated seeds and those that were soaked in water for 24 hours had the highest values for germination uncertainty. Mean germination time and synchronisation index showed no significant differences for all the pre-treatment methods. For all the traits measured, only germination uncertainty showed significant difference between the control and each of the pre-treatment methods. Germination was observed four weeks after incubation for all treatments and continued sporadically for nine weeks (figure 1). Soaking seeds in water for 48 hours gave the highest number of seeds that germinated during the first three weeks of incubation. De-husked and sand papered seeds did not germinate until the eighth and tenth week of incubation respectively. Our results show that soaking seeds in water stimulated germination of H. petersiana seeds (table 1), leading to a high germination percentage. This result suggests that dormancy in the seeds of H. petersiana may be classified as physical dormancy (Leubner, 2005). The duration of soaking influenced the germination capacity with low exposure time being more favourable compared to moderate and high exposure time. This result contradicted findings by Chin et al. (1988) working on Kentia palm seed and by Neto et al. (2014) working on Macaw palm in which soaking seeds in water did not improve germination for the entire duration of the experiment for the two palm species. In a separate study, Sanjeewani et al. (2013) demonstrated that seeds of Livistona rotundifolia (Lam.) Mart. require little amounts of additional water from outside to effect germination unlike those of H. petersiana. This suggests that palms exhibit different dormancy mechanisms across species which may necessitate a unique dormancy breaking mechanism for each particular species. It was, however, noted that imbibing seeds for extended periods resulted in more synchronised germination (table 1) although not significantly different from other treatments.
380 GERMINATION AND PHYSIOLOGICAL DORMANCY IN HYPHAENE PETERSIANA
60 Control De-husked
H2O soak 12 hours
40 H2O soak 24 hours
H2O soak 48 hours
H2SO4 10 minutes Potassium nitrate 1 hour Sandpapered
Germination (%) 20 Thiourea 1 hour
0
1 2 3 4 5 6 7 8 9 Incubation time (weeks)
Figure 1. Cumulative germination of Hyphaene petersiana seeds subjected to different pre-treatment methods.
Mechanical scarification involves any action that breaks the pericarp to allow water and gaseous exchange in the seed (Fransen et al., 2015). Sand papering and de-husking resulted in the lowest germination percentages. Germination was also slow and took nine and eleven weeks to be observed for sand-papered and de-husked seeds, respectively (figure 1). This is consistent with findings by Ferreira and Gentil as reported by Neto et al. (2014) who showed that removal of the endocarp from palm seeds has a high risk of damaging the endosperm and embryo. In terms of cumulative germination percentage, the two scarification methods were significantly different from the control and from each other. This suggest that H. petersiana seed germination is negatively affected by mechanical scarification. The effects of chemical treatment with thiourea and potassium nitrate did not differ from that of the untreated seeds (table 1) although they were associated with higher germination percentages (figure 1). Mean germination time and germination synchronisation in H. petersiana seeds is not affected by pre-treatment method (table 1). Although acid scarification has proved to be effective in improving germination of seeds in species with hard seed coats (von Fintel and Pammenter, 2004; González-Benito et al., 2006), in this study, the use of sulphuric acid resulted in no germination. This could have been due to the deleterious effects of sulphuric acid on both germination speed and percentage as noted by Neto et al. (2014). Sulphuric acid works by damaging the surface of the seed thereby allowing water and gas to enter and stimulate germination (Fransen et al., 2015). However, Fransen et al. (2015) highlighted that the concentration of the acid and duration of application are important factors controlling the efficacy of this dormancy breaking method. We, therefore, recommend that various sulphuric acid concentrations and duration of application be tested in order to get more conclusive results.
381 P. MUTETE, U. MAZARURA, E. GASURA, S. RICHARDSON-KAGELER, E. NGADZE AND S. KATIVU
Acknowledgements
“This work was conducted within the framework of the “Research Platform Production and Conservation in Partnership” (www.rp-pcp.org)”. This document has been produced with the financial assistance of the European Union. The contents of this document are the sole responsibility of the authors and can under no circumstances be regarded as reflecting the position of the European Union.
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