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Morphology, Storage Behavior, Dormancy, Germination Pattern and Chemical Composition of Tabernaemontana pandacaqui Lam. Seeds
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First Online 22-08-2022|
Background: Seed characteristics of Tabernaemontana pandacaqui Lam., an indigenous medicinal plant species from the Philippines, were investigated to help identify appropriate conservation strategies.
Methods: Mature seeds were tested for viability at different dormancy-breaking treatments and storage conditions. Seeds were also characterized morphologically and the seedling developmental pattern was established using the Biologische Bundesanstalt, Bundessortenamt and CHemische Industrie (BBCH) scale. We also determined the preliminary chemical composition through qualitative phytochemical screening and proximate analysis.
Result: Seeds of T. pandacaqui are obovate in shape with a 1000-seed weight of 20.99±0.62 g at 4.7% moisture content. Removal of the aril from the fresh seeds improved germination from 30%±0.98 to 84%±0.71 and shortened the time to seedling emergence from 45 days to 4 days after sowing. The seeds can tolerate drying to 4.7% moisture content and storage at -10°C for 3 months; thus, they have orthodox storage behavior. The germination pattern is epigeal phanerocotylar type, wherein the foliaceous cotyledons are raised (BBCH 09) and unfolded above the soil surface (BBCH 10). The seedling then develops an ovate to elliptic-shaped first true pair of leaves (BBCH 12) completing the germination process. The pre-dried seeds also have a high oil content of 28.7407±0.09316% with a strong presence of alkaloids and saponins. This indicates that the seed is amenable to ex-situ conservation through seedbanking, but its viability must be monitored in shorter intervals due to its high oil content.
T. pandacaqui is mainly propagated through seeds. However, information on seed morphology and germination pattern remains fragmentary. In addition, no record has been found on the seed storage behavior and chemical composition of T. pandacaqui (Chua and Horsten, 2001; Royal Botanic Gardens Kew, 2019). Data on its seed storage behavior and chemical composition are fundamental in developing appropriate conservation and management strategies (Hong et al., 1996). Furthermore, detailed information on its germination pattern and dormancy-breaking technique is important in establishing germination techniques and nursery management. Given the medicinal potential of T. pandacaqui, the study aimed to characterize the seed morphological features, evaluate the storage behavior, establish the initial dormancy-breaking technique, elucidate the detailed germination pattern and determine its preliminary chemical composition for genetic resource conservation and management.
MATERIALS AND METHODS
Mature seeds of T. pandacaqui (GB 67966) collected from plants maintained at the Crop Breeding and Genetic Resources (CBGR) Laboratory, Institute of Crop Science (ICropS), College of Agriculture and Food Science (CAFS), University of the Philippines Los Baños (UPLB) were used. Clean, undamaged and undeformed fruits and seeds were utilized for characterization. Seeds were sterilized by soaking in 5% sodium hypochlorite for five minutes and subsequently rinsed with distilled water prior to subjecting to dormancy-breaking treatments and seed storage behavior experiment.
Fruit and seed characterization
Morphological features of ten randomly selected mature fruits were observed. The number of seeds per fruit was recorded. Ten randomly selected seeds were used to measure and observe the shape, color and size (length, width, thickness). The weight of eight 100-seed replicates was used to determine the 1000-seed weight. The 1000-seed weight was computed using the formula:
To determine the dormancy behavior and germination percentage, clean and sterilized seeds were subjected to six treatments at room temperature: (1) control, seeds with aril; (2) removal of arils (3) scarification using sandpaper; (4) presoaking in 0.2M KNO3 for 12 hours; (5) presoaking in 100ppm GA3 for 12 hours; (6) presoaking in distilled H2O for 12 hours. For each treatment, two replicates of 100 T. pandacaqui seeds were sown in sterilized sand. Seeds with approximately 2 mm radicle emergence were considered germinated.
Seed storage behavior
A modified method to determine seed storage behavior, based on Hong and Ellis (1996), was used. Three replicates of 100 fresh seeds were tested for germination after the following storage conditions: (1) fresh seeds at 14.35% moisture content (MC) in ambient temperature, (2) slow-dried seeds at 4.7% MC and stored in -10°C for 3 months. Seed moisture content was determined using the modified International Seed Testing Association (ISTA) low constant temperature oven method.
Following the protocol of Gentallan et al., (2018), the detailed germination pattern of T. pandacaqui was elucidated based on the extended Biologische Bundesanstalt, Bundessortenamt and CHemische Industrie (BBCH) scale by Hack et al., (1992). Thirty T. pandacaqui seeds from the standard germination set-up were identified and observed for growth and development. The BBCH scale of the growth stages is represented by a two-digit code using numbers from 0 to 9 in ascending order. The first digit signifies the principal growth stage while the second digit code is the secondary growth stage.
Preliminary phytochemical analysis
Seeds of T. pandacaqui were pre-dried at 50°C for 24 hours, manually ground and pulverized using mortar and pestle and passed through an 18-mesh sieve. The powdered sample was stored in an air-tight container until further use.
The extraction process was carried out following the method described by Doctor and Manuel (2014) with modifications. Approximately 10 grams of the powdered seeds were macerated in methanol at room temperature using a laboratory blender and then suction filtered to collect the seed extract. This was repeated twice. The crude extract was then concentrated at 50°C and stored under refrigerated conditions until further use.
Qualitative phytochemical analysis of the crude extract was determined following the method with modifications.
Approximately 0.5 g of seed extract was diluted in distilled water and mixed with 5 mL of 0.1 N HCl solution in a hot water bath and filtered while hot. The filtrate was then added with 2-3 drops of Wagner’s reagent. The formation of a reddish-brown precipitate indicates the presence of alkaloids.
The crude extract was diluted in 0.1 N NaOH solution using a 1:1 (v/v) extract-NaOH solution ratio. The development of yellow color indicates the presence of flavonoids.
Ten milliliters of distilled water was added to 0.5 mL seed extracts in a test tube and shaken vigorously. The formation of stable froth that persists for 5-10 minutes indicates the presence of saponins.
Two to three drops of the crude extract were diluted in 1 mL of distilled water and the resulting solution was clarified through filtration. The filtrate was then added with a few drops of 1% ferric chloride solution. Development of bluish to black color indicates the presence of tannins.
The proximate composition (moisture content, ash, crude fiber, crude fat, crude protein and total carbohydrates) of pre-dried powdered seed samples was determined using the standard methods described by A.O.A.C (2000).
Analysis of Variance (ANOVA) and pairwise mean comparison using the Least Significant Difference (LSD) test was used to determine statistically significant differences between means of the dormancy-breaking treatments at α = 0.05. Germination (%) data were transformed to the arcsin of the square root of the fraction value before subjecting them to ANOVA.
RESULTS AND DISCUSSION
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.
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.
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.
CONCLUSION AND RECOMMENDATION
Conflict of interest
- Abe, F., Yamauchi, T., Guevera, B.Q. (1993). Indole alkaloids from Tabernaemontana pandacaqui in the philippines. Biochem Syst Ecol. 21: 847-848. doi:10.1016/0305 1978(93)90097-b.
- Association of Official Analytical Chemist (AOAC). (2000). Official Methods of Analysis: International 17th Edition. Gaithersburg, MD, USA, Association of Official Analytical Chemist.
- Blasco, F.A., De Guzman, G.Q., Alejandro, G.J.D. (2014). A survey of ethnomedicinal plants in surigao del sur mountain range, philippines. Int J Pure Appl Biosci. 2: 166-172.
- Blumenthal, A., Lerner, H.R., Werker, E., Poljakoff-Mayber, A. (1986). Germination preventing mechanisms in Iris seeds. Ann Bot 58: 551-561.
- Bradford, K.J. (2004). Seed Storage and Longevity. In: Seed production and quality. UC Davis, Seed Biotechnology Center, USA, pp 76-84.
- Boonchuay, W., Court, W.E. (1976). Alkaloids of Alstonia scholaris from Thailand. Planta Med. 29: 380-390. doi: 10.1055/s- 0028 1097681.
- Chua, L.S.L., Horsten S.F.A.J. (2001). Tabernaemontana divaricata (L.) R.Br. ex Roem. and Schult. In: [Van Valkenburg, J.L.C.H., Bunyapraphatsara, N. (eds)] Plant Resources of South-East Asia No 12(2): Medicinal and Poisonous Plants 2. PROSEA Foundation, Bogor, Indonesia.
- Colombo, R.C., Costa, D.S., Carvalho, D.U., Cruz, M.A., Roberto, S.R. (2018). Methods of aril removal and lightness conditions on seeds physiological quality of sour passion fruits. Braz J. Biol. 79: 404-409.
- Copeland, L.O., McDonald, M.F. (2012). Seed Storage and Deterioration. In: Principles of Seed Science and Technology. Springer Science and Business Media.
- De Vogel, E.F. (1979). Morphological types in dicot seedlings, with reference to their origin. Bull Soc Bot Fr. 126: 173-182. doi:10.1080/01811789.1979.10826406.
- Dey, A., Mukherjee, A., Chaudhury, M. (2017). Chapter 10-Alkaloids from apocynaceae: origin, pharmacotherapeutic properties and structure-activity studies. Stud Nat Prod Chem. 52: 373-488. doi:10.1016/B978-0-444-63931-8.00010-2.
- Doctor, T.R., Manuel, J.F. (2014). Phytochemical screening of selected indigenous medicinal plants of tublay, benguet province, cordillera administrative region, philippines. Int. J. Sci. Res. 4(4).
- Eckey, E.W. (1954). Vegetable Fats and Oils. American Chemical Society Monograph Series no. 123. Reinhold Publishing Corporation, New York.
- Ellis, R.H., Hong, T.D., Roberts, E.H. (1985). Handbook of Seed Technology for Genebanks (Volume I): Principles and Methodology. International Board for Plant Genetic Resources, Rome, Italy.
- Endress, M.E., Bruyns, P. (2000). A revised classification of the Apocynaceae. Bot Rev. 66: 1-56. doi:10.1007/BF02857781.
- Gentallan, R.P., Altoveros, N.C., Borromeo, T.H., Endonela, L.E., Morales, A.C. (2018). Seed morphology, storage behavior and germination pattern of Atuna racemosa. Seed Sci Technol. 46: 483-487. doi: 10.15258/sst.2018.46.3.05.
- Gruyal, H.A.A., Medina, T.F.T. (2019). Antibacterial activity and cytotoxicity against MCF-7 breast cancer cell lines of Tabernaemontana pandacaqui lam. (Apocynaceae). The Thailand Natural History Museum Journal. 13: 43-58.
- Hack, H., Bleiholder, H., Buhr, L., Meier, U., Schnock Fricke, U., Weber, E., Witzenberger, A. (1992). Einheitliche codierung der phanologischen entwicklungsstadien mono-und dikotyler Pfl anzen-Erweiterte BBCH-Skala, allgemein. [Uniform coding of the phenological development stages of mono- and dicotyledonous plants-Extended BBCH scale, general]. Nachrichtenbl Deut Pflanzenschutzd. 44: 265-270.
- Hong, T.D. (1996). Seed Storage Behavior: A Compendium. International Plant Genetic Resources Institute, Rome, Italy.
- Hong, T.D., Ellis, R.H. (1996). A Protocol to Determine Seed Storage Behavior. International Plant Genetic Resources Institute, Rome, Italy.
- International Seed Testing Association (ISTA). (2021). International rules for seed testing 2021. The International Seed Testing Association (ISTA), Bassersdorf, Switzerland. doi: 0.15258 /istarules.2021.02.
- Jordaan, A., Wessels, D.C.J. (1999). The aril of Colophospermum mopane: Its role during seed germination and fruit opening. S. Afr. J. Bot. 65: 392-397. doi: 10.1016/S0254-6299(15) 31028-0.
- Lange, A.H. (1961). Effect of the sarcotesta on germination of Carica papaya. Bot Gaz. 122: 305-311.
- Mahadimane, P.V., Chandra, S.S. (2020). Analyzing the phytochemical, anti-ulcer, anthelmentic and antioxidant potentials of Tabernaemontana dichotoma Roxb. ex Wall seed extracts. J. Nat. Remedies. 20: 89-95. doi: 10.18311/jnr/2020/24346.
- Merrill, E.D. (1926). An enumeration of Philippine flowering plants Bureau of printing. (Vol. 4).
- Padley, F.B., Gunstone, F.D., Harwood, J.L. (1994). Occurrence and Characteristics of Oils and Fats. In: The Lipid Handbook, [Gunstone FD, Harwood JL, Padley, FB (eds)] 2nd edn. CRC Press. Pp: 47-146.
- Perez-Harguindeguy, N., Díaz, S., Garnier, E. (2016). Corrigendum to: New handbook for standardized measurement of plant functional traits worldwide. Aust J. Bot. 64: 715-716. doi: 10.1071/BT12225_CO.
- Rao, N.K., Hanson, J., Dulloo, M.E., Ghosh, K., Nowell, A. (2006). Manual of Seed Handling in Genebanks (No. 8). Bioversity International, Rome, Italy.
- Rajjou, L., Debeaujon, I. (2008). Seed longevity: survival and maintenance of high germination ability of dry seeds. C. R. Biol. 331: 796-805. doi:10.1016/j.crvi.2008.07.021.
- Roberts, E.H. (1973). Predicting the storage life of seeds. Seed Sci Technol. 1: 499-514.
- Rodriguez, S., Vargas, I., Hijuelo, A., Loumeto, F., Silva, J.J., Pérez, J., Quirino, A., Fonseca, Y., Gomez, Y., Baldoquin, M., Oliva, D. (2019). Analysis of the effect of scarification process on papaya (Carica papaya Lin.) seeds germination. IntechOpen. doi:10.5772/intechopen.88012.
- Rolston, M.P. (1978). Water impermeable seed dormancy. Bot Rev. 44: 365-396.
- Royal Botanic Gardens Kew. (2019). Seed Information Database (SID). Version 7.1. Available from: http://data.kew.org/ sid/ (retrieved June 2019).
- Stevenson, D.E., Hurst, R.D. (2007). Polyphenolic phytochemicals- just antioxidants or much more?. Cell Mol Life Sci. 64: 2900-2916.
- Taesotikul, T., Panthong, A., Kanjanapothi, D., Verpoorte, R., Scheffer, J.J.C. (2003). Anti- inflammatory, antipyretic and antinociceptive activities of Tabernaemontana pandacaqui Poir. J Ethnopharmacol. 84: 31-35. doi: 10.1016/S0378-8741(02) 00264-7.
- Treutter, D. (2006). Significance of flavonoids in plant resistance: A review. Environ Chem Lett. 4: 147-157. doi: 10.1007/ s10311-006-0068-8.
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