Effect of scari cation and soaking on seed germination and viability of Podocarpus henkelii and P. latifolius (Podocarpaceae)


 Seed dormancy defines the environmental conditions in which a seed can germinate. Germination is timed to avoid unfavourable environmental conditions for plant establishment and growth. Podocarpaceae have an underdeveloped embryo which may delay germination. We aimed to establish if Podocarpus henkelii and P. latifolius produce underdeveloped embryos and examine the kind of dormancy the seeds have. The seed characteristics of P. henkelii and P. latifolius were observed, and we tested for seed germination after pretreating the seeds in water, hydrochloric acid and mechanical scarification and then tested for viability and the presence of the embryo. Embryos in fresh seeds of P. henkelii and P. latifolius had an embryo length/seed length ratio < 0.5 which suggested that the embryos were small and underdeveloped. Both podocarps produced fleshy seeds, P. latifolius had the smallest seed (mass: 0.19 g; length: 10.54 mm) and P. henkelii the largest (mass: 2.44 g; length: 23.69 mm). After exposing the seeds to the pretreatments, the seeds of both species did not germinate after ~6 months. However, viability testing showed that >90% of the seeds were viable with >95% of the seeds having embryos. Considering that the pretreatments did not facilitate seed germination in both species and that both podocarps have underdeveloped embryos that were viable P. henkelii and P. latifolius may have morphological or morphophysiological dormancy.


Introduction
Seed dormancy is the inability of a seed to germinate under environmental conditions that are favourable for germination, as such seed dormancy has evolved differently among taxa, allowing germination to occur when dormancy has been broken (Baskin and Baskin 2004;Fenner and Thompson 2005). Hence, a diverse range of dormancy mechanisms have evolved to ensure germination under suitable conditions. The most frequently used method for measuring seed dormancy is through observation of the absence of germination, therefore, seed dormancy can be observed as an all-or-nothing event where seeds can show maximum dormancy (all) and non-dormancy/germination (nothing). Seed characteristics play a vital role in meeting the requirements of seed germinations (Fenner and Thompson 2005). However, there are two important distinctions in seed responses which alter seed dormancy: (1) factors that are related to seasonal changes such as temperature and/or photoperiod which affect the sensitivity to other environmental factors (including light and water availability and relative humidity) and thus alter the level of seed dormancy, and (2) factors that immediately indicate that conditions are favourable for germination which can terminate dormancy and induce germination.
There are ve classes of seed dormancy, these include physiological (PD), morphological (MD), morphophysiological (MPD), physical (PY) and combinational (PY + PD) dormancy. In gymnosperms, physiological dormancy is the most dominant. Seeds experience different levels of PD ranging from embryos not growing, through to growing and producing normal seedlings (Baskin and Baskin 2004).
Morphologically dormant seeds tend to have underdeveloped embryos but are differentiated, these seeds are not necessarily dormant (physiologically) but require time to grow/mature before they can germinate.
Underdeveloped embryos are also evident in MPD but these seeds require dormancy-breaking treatment (e.g. strati cation). Mechanical or chemical scari cation is used to break PY dormancy as it makes the impermeable seed coat permeable to water; while PY + PD is evident in seeds that are impermeable to water combined with embryo dormancy (Baskin and Baskin 2004).
Most gymnosperms podocarps the female gametophyte is larger than the embryo (Ferrandis et al. 2011) which functions to bear gametes and protects and nourishes the embryo (Maseshwari and Singh 1967).
Podocarpaceae (podocarps) are reported to have underdeveloped embryos (MD) (Ferrandis et al. 2011;Chen et al. 2013). Several studies on podocarps have shown that germination time varies depending on species and/or environmental conditions required for seeds to germinate. Afrocarpus falcatus seeds required one-month moist storage and three weeks of incubation to reach germination (Negash 1992), while P. angustifolius seeds required >60 days to germinate when not soaked in water, and 35 days when soaked (Ferrandis et al. 2011). Podocarpus imbricatus and P. neriifolius germinated over a period of 16 -63 days and 20 -67 days, respectively (Ng 1992). Some seeds take a signi cantly longer to germinate, for example, P. nubigena did not germinate when on the surface in a canopy gap and only germinated to 22% after one year in the understory during a two-year experiment (Figueroa and Lusk 2001). Dodd and van Staden (1981) reported that to reach 68% germination P. henkelii seeds required ~160 days.
In this study, we determined the germination response of Podocarpus henkelii Stapf ex Dallim. & Jacks. and P. latifolius (Thunb.) R.Br. ex Mirb.. The objectives were to: (1) establish if embryo growth occurs in P. henkelii and P. latifolius seeds prior to germination, (2) measure seed germination, (3) assess if P. henkelii and P. latifolius seeds produce underdeveloped or fully developed embryos, (4) identify the kind of dormancy in these species and (5) measure seed morphological characteristics of P. henkelii and P. latifolius.

Methods
Seed collection: The species selected for this study were P. henkelii and P. latifolius. Podocarpus henkelii has a restricted distribution and occurs in the midlands of south-eastern South Africa which experiences summer rainfall and P. latifolius has a broad distribution and occurs in highland regions and in coastal temperate latitudes. During June 2019, freshly ripened P. henkelii and P. latifolius seeds were collected from Karkloof, Umgano and Mbona forest in the KwaZulu-Natal province, South Africa, and were combined to form a unique seed lot. The seeds were put into Ziploc bags, packed into cooler boxes at 0 -4 °C (Noel and van Staden 1976) and transported to the University of the Witwatersrand. Seed morphological traits: All seeds were tested for viability by submerging the seeds in tap water for 30 minutes; seeds that sunk were viable and were used for the experiment. To investigate seed morphology and embryo development in podocarp seeds fty fresh seeds of each species were cut open lengthwise with a razor blade and the seed length and width as well as embryo length and width were measured using a microscope.
Seed pretreatments and germination: Four hundred P. henkelii and P latifolius seeds were washed with tap water, pepped dry using a laboratory paper towel and at 1 °C until they were used for the experiment. The seeds were then washed with Sunlight liquid (a green detergent), rinsed four times with tap water to ensure that the detergent was completely removed, surface disinfected in 3.5% (w/v) sodium hypochlorite (NaOCl), and then rinsed four times using distilled water before they were placed in beakers. The seeds were divided into four treatments to break seed dormancy: (1) soaking in distilled water for 12 hr (control), (2) placed in a freezer in distilled water for 12 hr (cold strati cation), (3) acid scari cation by treating them with hydrochloric acid (HCl) of 50% concentration for 30 minutes and (4) mechanical scari cation (zester) and soaked in distilled water for 12 hr. After the seeds were soaked, imbibed seeds were introduced into petri dishes containing autoclaved and solidi ed agar at 15 g.l -1 (Puri ed agar, Sigma, South Africa). Seeds were inserted into the agar while leaving the micropyle exposed. Each treatment had 10 replicates with 10 seeds per petri dish, thus there were a total of 100 seeds per treatment for each species. Seeds were allowed to germinate in an incubator (Conviron plant growth chamber, model E8, UK) at 30 °C/15 °C (day/night), 12/12 hr (day/night) photoperiod and 50% relative humidity (Noel and van Staden 1976). Germination was recorded after seven days of incubation and then every two days for six months. Germination was de ned as the emergence of the radicle from the seed (Likoswe et al. 2008), where the radicle is more than 0.5 cm. The remaining seeds that did not germinate by the end of the experiment were tested for viability using a standard procedure tetrazolium test after exposing the embryo (International Seed Testing Association 2007).

Data analysis
Statistical analyses were conducted using the statistical program R ver. 4.1.1 (R Core Team 2021). Differences in the morphological characteristics of the seeds between species were tested using a student t-test. Since the viability percentages data violated the normality assumptions (Shapiro-Wilk test), even after arcsine transformation was applied, the Aligned Ranked Transformation procedure was used from the ARTool (Wobbrock et al. 2011;Elkin et al. 2021) R package. Means were separated using ART-C with a Tukey adjustment. Seed germination was not tested statistically as no seeds germinated during the experiment.
Interestingly, it is not known what disperses P. henkelii seeds, therefore we propose three hypotheses: (1) P. henkelii may be dispersed by gravity as the seeds are often seen rolling down slopes where they are abundant and seeds and seedlings are in close proximity with parent plants, (2) similar to Afrocarpus falcatus, P. henkelii may also be bird, bat, and/or monkey dispersed because they have similar appearances (Geldenhuys 1993;Negash 1995, Negash 2003, (3) seeds of P. henkelii may lack a dispersal agent since eshy fruit structures in podocarps evolved before modern dispersants emerged (Leslie et al. 2017) and (4) P. henkelii may have previously been dispersed by elephants which have since disappeared from their forests, however elephant remains have been found in their current distribution (pers. Obs.); this may explain why they have they have eshy fruits.
Podocarpus henkelii and P. latifolius embryos are linear-shaped and have a suspensor which links the embryo to the micropyle. The embryo length/seed length (E:M) in freshly harvested P. henkelii seeds was small (E:M = 0.37 + 0.01) as was in P. latifolius (E:M = 0.37 + 0.02); however, there was no signi cant different in E:M between P. henkelii and P. latifolius seeds (t = -0.330, df = 92.79, P = 0.7423) ( Table 1). An underdeveloped embryo is usually relatively small, has an E:M ratio < 0.5 and the embryo must grow within the seed before the radicle can emerge (Baskin et al. 2006a). We could not measure embryo length before the radicle emerged because none of the seeds germinated during the experiment after soaking in water, acid, and mechanical scari cation. After 189 days of germination assay, seeds that failed to germinate showed >90% viability in both species (Table 2). There was no signi cant difference in the viability of P. henkelii and P. latifolius seeds (F (1, 45) = 0.3049, P = 0.5836). In addition, the pretreatments did not have an effect on seed viability (F (1, 45) = 0.4054, P = 0.6692). Of the 97.67% P. henkelii and 93.33% P. latifolius seeds that were viable, the majority (97.33% and 98.33% of P. henkelii and P. latifolius seeds respectively) had differentiated embryos. Podocarps have been shown to have seeds with an underdeveloped embryo which grows to maturation as conditions improve (Baskin and Baskin 2005;Ferrandis et al. 2011;Chen et al. 2013). Underdeveloped embryos are considered as a primitive trait, although it can also be found in advanced angiosperms. This suggests that P. henkelii and P. latifolius undergo either MD or MPD because they both produce underdeveloped embryos that need time to grow before germination can occur. Finally, it is necessary to provide P. henkelii and P. latifolius seed embryos time to grow so that germination can occur.
After attempting to germinate P. henkelii and P. latifolius seeds for six months, no seeds germinated. Laughton (1938), Noel and van Staden (1976) and Dodd and van Staden (1981) germinated P. henkelii seeds within two -ve months of incubation. Geldenhuys (1993) reported 51% germination when seeds are soaked in hydrochloric acid, 2% germination when the seeds pass through bushpigs intestines and 71% germination when soaked in water. Podocarpus latifolius showed 86% germination after three months (Adie and Lawes 2009). Interestingly, Bussmann and Lange (2000) showed that P. latifolius seeds did not germinate after three months of germination and suggested that germination was affected by seed storage time. After following all protocols all seeds did not germinate, as such future studies should germinate seeds soon after collection, remove the sclerotesta, germinate seeds in soil instead of agar and germinate the seeds in greenhouse conditions instead of incubators.