Seed germination and seedling growth of Pycnanthus angolensis(Welw.)Warb.,African false nutmeg

Akinropo M.S.,Sakpere A.M.A.

Department of Botany,Faculty of Science,Obafemi Awolowo University,Ile-Ife,Osun State 230103,Nigeria

ABSTRACT This study investigated the germination behavior and seedling growth of Pycnanthus angolensis seeds.The germination study was carried out in the laboratory and included pretreatment studies and observation of the seed-germination process.For each treatment,three replications of 20 seeds were sown in a transparent plastic germination box(12cm×22cm×5cm)lined with moistened filter paper at room temperature.To monitor seedling growth,seedlings were transplanted into pots filled with topsoil and laid out in a completely randomized design.Ten seedlings replicated thrice were measured monthly;and the shoot height,leaf area,and root length,as well as the fresh and dry weights of the seedlings,were recorded.Results showed that mechanically scarified seeds exhibited the significantly highest germination percent(83.33%,P ＜0.005),followed by control seeds(70.83%),whereas seeds treated with 98%sulphuric acid(H2SO4)displayed the lowest germination percent(1.67).The endospermous seeds exhibited cryptogeal germination,while seeds stored for a month(with or without arils)failed to germinate.During seed germination,radicle protrusion continued with a pseudo-opening of the root,through which the brownish cotyledonary petiole was emitted,thus releasing the plumule at the posterior position.Moreover,P.angolensis exhibited a slow growth rate,attaining a shoot height of 73 cm within a year.The highest positive change in leaf number and area was recorded in the fourth month,a period during which the least change in shoot height occurred.The study concluded that mechanical scarification of the seeds ensured significant and faster germination than chemical scarification or no treatment at all.Additionally,P.angolensis displayed a cryptogeal germination,with the seedling growth of the tree species observed to be slow.

Keywords:cryptogeal germination;cotyledonary petiole;pretreatment;tree

1 Introduction

Pycnanthus angolensis,a tree species native to tropical Africa(GRIN,2008),is a member of the family Myristicaceae.The family Myristicaceae is a rich source of bioactive phytochemicals useful in treating various ailments(Ancolioet al.,2002;Dennyet al.,2008;La Frankie,2010).Pycnanthus angolensisis common in lowland rainforests and secondary forests,where it can become dominant.P.angolensishas a variety of human uses and is commonly called boxboard,cardboard,Kombo butter,wild African nutmeg,and African false nutmeg.In Africa,it is widely known as Ilomba(Richter and Dallwitz,2009);and it is called Akomu by the Yorubas in Nigeria.Morphologically,members of the Myristicaceae family are easily recognizable in the field by their straight trunks,characteristic bloodlike sap exudates,few insect-spotted leaves,and nutmeg like fruits that are considered one of the most primitive of the angiosperms(Katendeet al.,1995;Orwaet al.,2009).The seeds ofP.angolensisare homeohydrous,i.e.,recalcitrant(Babalola,2006;Dike and Aguguom,2010;Onefeli and Akinyele,2013),and are also black,with brightly red arils(Figure 1).They occur solitarily within the two valves,hence are dispersed mainly by animals and water(Babalola,2006).

Many tropical tree species have recalcitrant seeds(Berjak and Pammenter,2004;Baskin and Baskin,2005),and study of their seed germination has been limited(Mngombaet al.,2007).Some studies on seed germination ofP.angolensishave been carried out.Mapongmetsemet al.(1999)reported that pretreatment by hand scarification of the extracted seeds ofP.angolensisis the most efficient treatment.They,however,did not explain if the arils were removed or left in place;it is assumed that,by the process of extraction,the arils were removed.It has also been reported that soaking the seeds in cold water for 24 hours hastens germination(Mapongmetsem,2007).In a study of thein vitrogermination ofP.angolensis,Bello and Akinyele(2016)reported that the seeds germinate better under priming than when sown directly.Oboho and Igharo(2017)showed that aril removal produced the best germination performance(83.33%),followed by soaking seeds for 48 hours(70%),and thatP.angolensisexhibited the hypogeal type of germination.Although Mapongmetsemet al.(1999)reported studies with sulphuric acid,they utilized only one concentration (98%) for 20 minutes, with 0% germination.There is a need to find out if lower concentrations and reduced exposure time can have a more beneficial effect.Preliminary studies also revealed that germination inP.angolensismay not be hypogeal in nature.

The present study describes the observations made on seed germination ofP.angolensis,as well as on its germinability.We also assess seedling growth parameters in order to provide the basic information needed for use in forest-restoration programmes.

2 Materials and methods

Seeds ofPycnanthus angolensiswere collected from actively growing trees during the dry season(November through March)on the Obafemi Awolowo University campus, Ile-Ife, Osun State, Nigeria(7°31'8.4"N,4°31'15.96"E).Fresh seeds(collected often along with red arils and/or without arils)and stored seeds,were used throughout the germination experiments.

2.1 Germination-improvement studies

The experiments were carried out on two sets of seeds—fresh seeds(i.,with arils;and ii.,without arils)and stored seeds(i.,with arils;and ii.,without arils)for 4 weeks.Germination-improvement studies were carried out by chemical scarification,using concentrated sulphuric acid(H2SO4)—50%for 40 minutes,50%for 20 minutes,and 98%for 2 minutes and mechanical scarification(using sandpaper).Fresh untreated seeds(without arils)served as the control.The germinability of fresh seeds with arils and stored untreated seeds(with and without arils)was also investigated.The untreated and pretreated seeds were then sown in transparent plastic boxes and kept in a laboratory at ambient conditions.Twenty seeds were planted in three replicates and repeated three times per treatment;the seeds were watered(10 mL)at an interval of 2 days.Counts of germinated seeds were made at an interval of 2 days for 30 days.Visible protrusion of the radicle through the seed coat was taken as the criterion for germination.

2.2 Description of germination behavior

The fresh seeds(without arils)were washed and surface sterilized with 10% sodium hypochlorite(3.85%m/v)for 15 minutes.The seeds were further rinsed, with four changes of distilled water, after which they were sown in petri dishes lined with Whatman No.1 filter paper.The petri dishes were left inside a laboratory at room temperature and watered every other day with distilled water.Daily observations,including radicle protrusion and plumule emergence,as well as shoot elongation,were documented.

2.3 Seedling growth

To assess seedling growth parameters,140 seedlings ofP.angolensisfrom fresh seeds were separately transferred after 2-3 weeks to topsoil in pots(28 cm in diameter and 12 cm in depth)with perforation at the bottom to allow for drainage.The seedlings in each pot were allowed to grow for 4 weeks to allow their establishment,after which the various growth parameters were measured monthly for 12 months.Thereafter,the change in growth per month was calculated and the values used to draw a line graph.

2.4 Measurement of growth parameters

Using thread and a meter rule,the shoot height of the seedlings was measured as the distance from the soil level to the tip of the terminal bud of the plant.

The leaf area was calculated using the modified Hoyt and Bradfield(1962)formula,as follows:

where L represents leaf length measured as the distance between the base of the leaf and the leaf's apex;W represents the width of the leaf,measured as the most expanded portion of the leaf,using a meter rule;and C.F=0.68,which represents the correction factor for the shape of the leaf.

The seedlings were uprooted carefully and separated into shoots and roots.The washed roots were mopped dry with blotting paper and weighed using a Mettler Toledo Weighing Balance.The shoots and roots were then dried in an oven at 80°C for 48 hours and weighed to determine the dry weight.

2.5 Statistical analysis

The germination data were subjected to an analysis of variance using SAS Software(SAS 9.1.2;SAS Institute Inc.,Cary,NC,USA).Differences between means of the recorded parameters were separated using Duncan's multiple range test(DMRT)at the 5%probability level.

3 Result

For germination-improvement studies,fresh seeds ofP.angolensisstarted germinating on days 4,8,8,12,and 22 of planting for mechanically(sandpaper)scarified seeds,the control(untreated seeds),seeds treated with 50%H2SO4for 20 minutes,seeds treated with 50%H2SO4for 40 minutes,and seeds treated with 98%H2SO4for 5 minutes,respectively(Table 1).The highest germination rate was exhibited by the mechanically scarified seeds(2.97),followed by the control seeds(2.53),while the lowest germination rate was recorded for seeds treated with 98% H2SO4(0.075).Freshly collected seeds with arils and stored seeds(with or without arils)as well as those treated mechanically and chemically,failed to germinate.The seeds ofP.angolensiswere observed to be elliptical,with a yellowish furrow on the ventral face,and endospermous in nature.The hard endosperm(almost completely filling its inner part)surrounds the centrally positioned lamina-shaped embryo(Figure 2).Observations of germination behavior revealed thatP.angolensisshoots emerged by 14-16 days after germination.Although the germination ofP.angolensisresembles the normal hypogeal method,it was highly modified;instead of the epicotyl elongating to carry the plumule above the ground,the plumule emerged from a cotyledonary petiole below the ground,exhibiting cryptogeal germination.The seed germinated by the protrusion of a yellow-coloured pseudo radicle,which soon elongated to reveal the whitish true-root region.This true root extended up to about 6 cm in length before the emergence of a brownish portion,the cotyledon petiole,within 10 days after germination(Figures 3a-3d).Thereafter,there was a visible swelling(i.e.,the portion bearing the shoot)at an extension,called the cotyledon petiole,beyond the root portion(Figure 3e).This swelling split open within 10-15 days,forming a cotyledonary node,allowing for the gradual emergence of the shoot from the cotyledon petiole after 24 days,before the negative geotropic growth of the shoot(Figures 3f-3h,4a-4d).

Table 1 Effect of different seed pretreatments on germination of fresh Pycnanthus angolensis seed

Figure 2 The longitudinal section of Pycnanthus angolensis seeds:(a)before germination;both(b)and(c),after germination

3.1 Growth-rate measurement

The mean shoot height ofP.angolensisincreased from 4.98 cm in the first month to 73.26 cm in the twelfth month.Also,the monthly changes in the shoot height during the 12-month period were slightly different from one another.The change in shoot height during the first 3 months was more or less constant,higher than that of the fourth month but lower than that of the fifth month,corresponding to the period during which the seed coat bearing the cotyledon dehisced from the seedlings.Figure 5 shows 5-monthold seedlings ofP.angolensis.

The leaf area increased from 104.60 cm2when seedlings were 2 months old to 1,065.36 cm2when they were 12 months old.Figure 6 shows the mean total number of leaves and leaf area ofP.angolensisduring the period of investigation,from the eighth week to the twelfth month.As shown in Figure 6,the maximum change in leaf area(324.6 cm2)and the highest mean change in number of leaves were observed in the fourthmonth,a period prior to the cotyledons'dropping off the seedlings(i.e.,the fifth month).

Figure 3 Germinating seeds of Pycnanthus angolensis shown at(a)day 4,(b)day 6,(c)day 7,(d)day 8,(e)day 21,(f)day 23,(g)day 25,and(h)day 30

Figure 7 shows the mean change in shoot height and root length over a period of 12 months.The root length ofP.angolensisincreased from 4.97 cm in week 4 to 73.40 cm in the twelfth month.The highest change in root length was recorded in months 2,11,and 12,respectively.

The fresh weight of theP.angolensisshoot increased gradually,from 2.12 g in the first month of growth to 52.07 g by the end of a year,whereas the fresh weight of the root increased from 0.29 g at the end of week 4 to 30.73 g by the end of a year(Figure 7).

Additionally,the total plant weight increased from 2.48 g in the first month to 82.79 g in the 12-monthold seedlings,with the highest change observed in the fifth month(Figure 8).The highest change in shoot dry weight occurred in the ninth month(Figure 9).In contrast,the highest changes were recorded for the root dry weight in the fifth month,which increased slightly thereafter to the end of the twelfth month.The change in total plant weight varied across the months;however,the first major increase occurred in the fifth month.

Figure 4 Germinating seeds of Pycnanthus angolensis shown at(a)day 32,(b)day 34,(c)day 38,and(d)before transplanting,respectively

Figure 5 Five-month-old seedlings from freshly collected Pycnanthus angolensis seeds

Figure 6 Growth rate of Pycnanthus angolensis seedlings(a)with respect to the number of leaves and the total leaf area and(b)the change in growth rate with respect to the number of leaves and the leaf area

Figure 7 Change in the growth rate of Pycnanthus angolensis seedlings with respect to shoot height and root length from the first month to the twelfth

4 Dicussion

Ready germination of freshly collectedP.angolensisseeds(i.e.,with fresh arils removed)without pretreatment and the lack of germination in seeds stored for a month are an indication that the viability of seed is greatly reduced with storage.Because the seeds are reported to be recalcitrant(Dike and Aguguom,2010;Onefeli and Akinyele,2013),the lack of germination of stored seeds could be due to recalcitrance,dormancy,and/or seed deterioration.The family Myristicaceae to whichP.angolensisbelongs has been reported to exhibit morphological dormancy (Baskin and Baskin,2005).The early germination of the control seeds proved the homeohydric nature of the seeds.Seeds harvested with dry arils did not germinate,indicating that the arils can also inhibit seed germination,which may be responsible for nonavailability of the seedling in its natural habitat. Oboho and Igharo(2017)also observed similar results with the same plant species and reported an extremely low percent of germination from the seeds ofP.angolensisplanted with arils and a low percent(＜40%)from stored seeds with the seed coat completely removed.Among chemically scarified seeds,the maximum percent of germination(11%)was recorded from seeds treated for 20 minutes with 50%sulphuric acid.TheP.angolensisseed coat is crunchy and hard;and the arils dry and become hard,which could affect their permeability to water and air.Application of 98%or 50%H2SO4either for 40 or 20 minutes affected the seeds and caused some kind of wet combustion of the seed coat.This result corroborates the work of Mapongmetsemet al.(1999)onP.angolensisseed pretreatments and germination.The authors reported 100%,79.2%,and 0%germination by seeds punctured at the ends,seeds under the control conditions,and seeds with acid scarification,respectively.Breaking dormancy of seeds,especially seeds with a hard seed coat,with concentrated sulphuric acid is well documented in literature(Baeset al.,2002;Tanaka-odaet al.,2009;Ayisire,2014;Purohitet al.,2015).Acid scarification has been reported to be effective for some tropical species such asAcacia nilotica,Prosopis juliflora,Sapindus trifoliatus,Annona senegalensis(Naiduet al.,1999),Enterolobium cyclocarpum,Prosopis africana(Ayisire,2014),andCyclocarya paliuru(Fanget al.,2006).Brahman(1996),however,reported that acid scarification was not applicable to all seeds,particularly easily permeable seeds,as the acid penetrates and damages the embryo.Scarification using 98%sulphuric acid was reported to damage the embryo ofParinari curatellifolia(Ming'omba,2007).Swaiet al.(2004)reported that nicking the seeds or completely removing the seed coat generated a higher germination percent,which is in line with our results.It appeared that the 98%H2SO4was too corrosive to the seed coat ofP.angolensis,rapidly corroding the seed coat and penetrating the seed to damage the embryo.Owing to this finding,it is suggested that very low concentrations of H2SO4should be tested for their effect on germination because the lowest concentration used in this study(50%)at the lowest time(20 minutes)had the highest percent of germination among the H2SO4-treated seeds.However,removal of the arils of the seeds is important for the fast and uniform germination ofP.angolensisseeds.

Figure 8 Change in the growth rate of Pycnanthus angolensis seedlings with respect to the fresh shoot weight,root weight,and total plant weight from the first month to the twelfth

The germination ofP.angolensisappears to follow the normal hypogeal germination process,but the germination is referred to ascryptogeal.This type of germination has been reported by Jackson(1974).Ugeseet al.(2005)and Chimsah(2015)also reported cryptogeal germination forVitellaria paradoxa, in which the shoot bud emerges from underground after 28 days.Cryptogeal germination produces an apparent radicle that carries the plumule below the soil surface prior to shoot elongation.This germination mechanism has been referred to as an adaptation to reduce water loss and is relatively common in the monocotyledons,as well as in a number of taxonomically unrelated families of herbaceous and woody dicotyledons(Clarkson and Clifford,1987;Burrowset al.,1992).Cryptogeal germination could follow either epigeal or hypogeal germination.Jackson(1974)reported cryptogeal germination in species ofCombretum(C.molle,C.fragrans,C.binderianum),which in the first stage follows apparently a normal epigeal germination. However, germination inCombretum sericeumandCombretum paniculatumresembles the normal hypogeal process,i.e.,the cotyledons are not withdrawn from the seed coats.Savanna tree species such asVitellaria paradoxa,Gardenia erubescens,Lophira lanceolata,Pterocarpus erinaceus, andPilostigma thonningiiwere also reported to undergo cryptogeal germination(Jackson,1974).Burrowset al.(1992)opined that such a mechanism is an adaptive feature for Pyrophytes.P.angolensisfollows a normal mode of hypogeal germination,and its degree of cotyledon fusion is similar to that ofVitellaria paradoxa;but the radicle later splits open,forming cotyledon petioles,thereby releasing the shoots.Clarkson and Clifford(1987)concluded that the fusion of the cotyledons and large-seededness are the major characteristics associated with cryptogeal germination.Oboho and Ighalo(2017)reported hypogeal germination inP.angolensis.This report could be due to the fact that they planted the seeds directly into the soil and so could not observe the process of germination,unlike in the present study where the seeds were germinated in a transparent plastic box.

Figure 9 (a)Growth rate of Pycnanthus angolensis seedlings with respect to dry matter(shoot weight,root weight,and total plant weight)from the first month to the twelfth;(b)Change in growth rate with respect to dry matter(shoot weight,root weight,and total plant weight)from the first month to the twelfth

One year-old seedlings ofP.angolensiscan attain a height of approximately 73 cm, with about 35 leaves,a root length of 73 cm,and a total leaf area of 1,065 cm2.Pittoet al.(2004)expressed the view that this information is significant in light of the fact that growth and development studies constitute primary criteria for evaluating the success of forest-restoration efforts.From the results,P.angolensisis observed to be a slow-growing tree.

A greater increase in seedling growth ofP.angolensiswas observed after an increase in total leaf area,resulting in an increase in photosynthesis that provided more food for growth.The larger the total leaf area,the greater the surface exposed to light,resulting in an increase in food production,which is vital for growth.The root length increased by 14.84%in the second month,which is important for the establishment of the seedlings by ensuring an adequate supply of water and nutrients.Root length was the first parameter that recorded a very high change in growth from one month to the other.There was no increasing change in the shoot height by the fourth month,as the number of leaves and the leaf area were increasing.There was an 83%increase in leaf area between the second and third months of growth and a 169%increase in leaf area between the third and fourth months of growth.Early increase in leaf area may ensure early establishment by supplying metabolites needed for growth. Shoot height,root length,and weight parameters were all reduced by the fourth month,while the leaf area was increased. All available nutrient and photosynthates were partitioned towards increase in leaf area.Subsequently,in the fifth month,there was a significant increase in the growth of the different parameters,caused by the increase in the available photosynthates due to the increase in leaf area.

5 Conclusion

Chemical and mechanical scarification methods were used to investigate the germination behaviour ofP.angolensisseeds.The study allowed us to conclude that freshly collected seeds ofP.angolensiswithout arils are highly viable and that mechanical scarification is more appropriate for improving seed germinability.P.angolensisseeds exhibit cryptogeal germination,and the germination can be enhanced by removing the arils from the seeds.Further studies need to be carried out on the use of H2SO4for chemical scarification of the seeds.The tree is a slow-growing species,with the first rapid growth of the root length in the second month of growth;and the number of leaves,as well as the leaf area,records a maximum change in growth by the fifth month.

Acknowledgments:

The authors are grateful to the anonymous reviewers for insightful comments that have improved the manuscript significantly.