Fruit and Seed Characteristics
Tabernaemontana pandacaqui bears smooth to hairy, orange to red-orange capsules at maturity. The fruit is composed of two lateral capsules containing an average of 56 brown-colored seeds that are fully enclosed by a vivid reddish-orange aril. Seeds are obovate in shape with respect to the point of radicle emergence. The surface is wrinkled with longitudinal grooves and the aril is attached to the deep hilar groove. Seeds are relatively small being 6.92±0.19 mm in length, 4.69±0.18 mm in width, 3.16±0.17 mm in thickness with a 1000-seed weight of 20.99±0.62 g at 4.7% moisture content. It has ruminated endosperm corresponding to the description of
Endress and Bruyns (2000) of
Tabernaemontana species.
Seed dormancy
De-arillated
T.
pandacaqui seeds started germinating 4 days after sowing (DAS) while arillated seeds were observed to have induced dormancy with non-uniform germination starting at 45 DAS until 90 DAS. Germination of arillated seeds (30%) was significantly lower compared to de-arillated seeds with 84% germination. Scarification of seeds using sandpaper improved the germination of
T.
pandacaqui from 84% to 94%. Presoaking treatments of de-arillated seeds in 100 ppm GA3, distilled water and 0.2 M KNO3 did not significantly increase the germination of
T.
pandacaqui with 91%, 89% and 88% germination, respectively (Table 1). This demonstrates that the arils of the seeds must be removed to ensure a higher germination rate during regeneration or propagation. Unlike in nature where the aril creates favorable moisture conditions such as in the case of
Colophospermum mopane (Jordaan and Wessels, 1999), arils may contain inhibitory substances that delay germination
(Lange, 1961; Rodriguez et al., 2019). Aside from this, arils may prevent water imbibition by acting as “plugs”
(Rolston, 1978) and arillated seeds were also susceptible to pathogen infestation during viability testing. Aril removal or scarification increases seed germination rates such as in the case of
Iris spp.
(Blumenthal et al., 1986) and
Passiflora spp.
(Colombo et al., 2018).
Seed storage behavior
Freshly extracted mature de-arillated seeds have a moisture content of 14.35% and seed viability of 90%. Seeds that were slow-dried to 4.7% MC and stored at low temperature (-10°C) for three months have comparable seed viability of 87%. Since the seeds can tolerate drying up to 4.7% and freezing up to -10°C for three months, the seeds were considered to be desiccation-tolerant and were categorized as having orthodox storage behavior
(Roberts, 1973). This is the first report on the seed storage behavior of
Tabernaemontana pandacaqui Lam.
; however, plants on the same genus,
Tabernaemontana coffeoides Bojer ex A.DC.,
Tabernaemontana elegans Stapf and
Tabernaemontana pachysiphon Stapf were documented to have orthodox seeds
(Royal Botanic Gardens, Kew, 2019). The character of
T.
pandacaqui seeds concurs with the general characteristics of an orthodox seed,
i.
e. smaller size and lower initial moisture content than recalcitrant seeds (>50%) at physiological maturity
(Copeland and McDonald, 2012). Orthodox-seeded species are long-lived
(Roberts, 1973) and can be stored long-term at about -18°C and 5% moisture content
(Ellis et al., 1985). Hence,
T.
pandacaqui can be conserved in seed genebanks. Moreover, further ultra-drying experiments could yield cost-efficient conservation strategies of
T.
pandacaqui seeds.
Seedling development
The germination pattern of
T.
pandacaqui seeds was explained by germination (stage 0) and leaf development growth stages (stage 1) of the BBCH scale (Table 2). Radicle emergence of de-arillated fresh
T.
pandacaqui seeds was observed as early as 4 DAS (BBCH 05). The radicle elongates and develops root hairs and lateral roots (BBCH 06). Hypocotyl breaks through the seed coat (BBCH 07), elongates forming the hypocotyl arch (BBCH 08) and subsequently emerges above the soil surface with the cotyledons remaining inside the seed testa (BBCH 09) (Fig 1). Ovate foliaceous cotyledons emerged from the testa (BBCH 10) that were followed by the appearance of leaf primordia that developed into the first true pair of leaves (BBCH 12). The first true leaves are ovate to elliptic in shape and glabrous with smooth and entire margins. The seedling development pattern of
T.
pandacaqui exhibits the usual phanerocotylar epigeal type of germination with foliaceous cotyledons that have photosynthetic activity
(Perez-Harguindeguy et al., 2016). This species exhibits the
Macaranga type of seedling development wherein the paracotyledons are exposed and raised above the ground
(De Vogel, 1979). Its characteristic non-plastic germination pattern indicates that positioning studies during planting can be undertaken to ensure seedling establishment.
Preliminary chemical composition
This is the first report of the proximate and phytochemical composition of
T.
pandacaqui seeds. The phytochemical screening showed the presence of alkaloids, flavonoids and saponins on the methanolic extracts of
Tabernaemontana pandacaqui seeds (Table 3). The presence of these phytochemicals is consistent with the methanolic extracts of
Tabernaemontana dichotoma Roxb, seeds
(Mahadimane and Chandra, 2020). The presence of flavonoids, however, can increase seed longevity. Having antimicrobial properties, flavonoids, particularly when localized in the seed coat, may provide chemical protection against pathogen infection
(Treutter, 2006). Similarly, alkaloids may also provide this effect
(Dey et al., 2017; Gruyal and Medina, 2019). Flavonoids can also promote longevity in storage by scavenging reactive oxygen species inducing cellular level protection as an antioxidant, such as in the case of
Arabidopsis (Stevenson and Hurst, 2007; Rajjou and Debeaujon, 2008). This further supports the storability of
T.
pandacaqui seeds in seed genebanks.
On the other hand, the proximate composition of its seeds denotes high oil content of 28.7407±0.09316% (Table 4) which is higher than soybean with an oil content of around 19.56%
(Eckey, 1954; Paddley et al., 1994). Seeds with high oil content are often observed to have lower longevity in storage among orthodox types and should be monitored for viability after five years in long-term storage conditions
(Rao et al., 2006). Thus, although its seed is amenable to
ex-situ conservation through seedbanking, its viability must be monitored in shorter intervals due to its oily characteristic. Conservation and management practices specific for storing oily orthodox seeds should be followed. This includes following the low-constant oven drying method in routinely determining the seed moisture content
(Rao et al., 2006; ISTA, 2021). Oily seeds tend to have lower equilibrium moisture levels at a specific relative humidity and temperature compared to starchy seeds
(Bradford, 2004). In seed viability equations, species-specific moisture content constants (Cw) in oily seeds were much lower
(Hong et al., 1996). This means that seeds of
T.
pandacaqui will dry to lower moisture contents, at the same relative humidity and temperature, compared to other non-oily species; however, it is necessary to dry these oily seeds more to obtain the same relative increase in longevity during storage.
