Germinating potential of Phyllanthus amarus seeds: evaluation of biochemical parameters
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Collection, authentication, optimisation of storage conditions and selection of suitable propagules for propagation and conservation are crucial in the case of wild-growing medicinal plants which have adaptive variations under different climate regimes. The present study aims to understand the factors (here, seed types and storage conditions) that influence the germination potential of wild-collected Phyllanthus amarus L. (Euphorbiaceae) using biochemical characteristics of seeds, besides taxonomical authentication of the species. The amplicons of 1150 bp in SCAR marker analysis and 6–7 ribs on seed surface shown by scanning electron micrograph confirmed that the wild-collected specimen is P. amarus. Interestingly, the mature green seeds harvested and shade-dried within 0–3 days exhibited maximum sink percentage (55.8%) and showed a significantly (p < 0.001) higher germination percentage with carbohydrate and proteins than the float seeds. However, the float seeds contained 33% less fat content than the sink seeds. Upon storage, the germinability of P. amarus seeds was found to be 12 months at 4 °C without moisture. This shows that viable seeds can be harvested and stored appropriately to ensure longevity. This study makes a case for the preservation of seasonal, short lifespan seeds such as P. amarus.
KeywordsPhyllanthus amarus seed Germinability Longevity Storage
The medicinal herb Phyllanthus amarus L. (Euphorbiaceae) is widely distributed in tropical and subtropical regions of Central and South Asia . It is one of the most essential ethnomedicinal herbs that is orally administered for jaundice, asthma, hepatitis and malaria, and is known to have diuretic, antiviral and hypoglycaemic properties . The whole plant extract is reported to contain alkaloids, flavonoids, phenols, coumarins, tannins, terpenoids and lignans are responsible for its curative properties . More than 60% of the people in the developing countries depend on the herbal drug for their ailments . Despite having medicinal uses, this medicinal plant is not cultivated, so it has been gathered from wild sources and its availability will be challenged by herbivorous overgrazing  and climatic change . Therefore, the species is likely to be extinct or loss of its habitat at a faster rate [7, 8], so its long-term conservation is an important criterion to be considered . Recently, researchers recognised the value of conserving the medicinal plant, but they are facing several challenges that hampered their efforts . As the wild-grown P. amarus has endured adaptive variation under different climate regimes,  germinability potential of seeds and its biochemical nutrients constituents under natural habitats remains a threatening reality for in situ conservation of the species.
Though the medicinal plant has been distinguished according to the morphological features by the taxonomist, the species P. amarus is often mistaken for other closely related species such as Phyllanthus niruri, Phyllanthus debilis, Phyllanthus maderaspatensis and Phyllanthus virgatus . In spite of taxonomic keys available for identification of P. amarus, many of the Phyllanthus species are invariably collected under the name P. niruri because all the species share a common Tamil vernacular name ‘Keezhanelli’. Thus, these species are used interchangeably by local collectors. . However, a number of publications from India use the name P. niruri, which actually is endemic to America, only . The P. niruri described in the Flora of British India  has been reported to be a mixture of three closely related but distinct species, namely P. amarus, P. debilis and Phyllanthus fraternus. Thus, it is not uncommon to find admixtures of related/allied species and sometimes also of other unrelated genera. The consequences of species admixtures can range from reduced bio-efficacy of the drug to lower the trade value as well as threats to the very safety of herbal medicines . Earlier studies have attempted to resolve the nomenclatural problem persisting with P. amarus for easy identification of the related species by analysing the morphological, anatomical characters and sequence-characterised amplified regions (SCAR) markers .
Most annual medicinal plants are seasonal and their availability primarily relies on viability and successful germination of seeds. However, the extent of seed viability is influenced by various environmental factors such as temperature, relative humidity, salinity, light and soil moisture. . Typically, the fresh seeds of P. amarus show less than 50% germination and the viability is found to decrease significantly with storage time . As low-quality seeds can reduce the seedling percentage and seedling emergence rate , the seeds need to be harvested at the right stage of maturity. More importantly, the seeds should be dried appropriately for storage [19, 20, 21]. The foremost problem is improper seed storage conditions such as low room temperature and adequate moisture can promote fungal growth  and insect development in seeds , which shortens seed viability . The standard method to overcome this problem is proper maintenance, which has been demanded the optimistic seed viability for a longer period that is very essential to conserve the species.
Seeds accumulate carbohydrates, proteins and lipids in different proportions depending on the species in a given condition. During seed development, the reserve material is utilised and weight is gained for them to sink in water. This phenomenon mainly depends on the assimilate level and amount of reserve material . The inherent moisture level will be the lowest in mature seeds and highest in the initial stage. Initially, reserve materials are loaded heavily with carbohydrates and lipids (non-polar), and in later stages of development, the lipids become negligible . Alpha-amylase and the accompanying hydrolytic enzyme action on the starch are the predominant form of glucose, which is then utilised for the growth of tissue, shoots and roots from the seeds .
Based on the above background, it has been observed that identifying and harvesting seeds at a suitable stage of development and drying and storing them affects the viability of the seeds, but there has been no study to address this problem in P. amarus so far. Therefore, this study has been conducted in order to find optimal seed storage conditions with reference to factors that limit the seed germinability of authenticated P. amarus by morphological and molecular basis.
2 Materials and methods
2.1 The study area, collection and identification of P. amarus seeds
Wild plants of P. amarus were collected from Nandhi Nagar, Dharmapuri District, India, which lies between 12° 6′ 23.4972″ N 78° 8′ 10.1076″ E at an altitude of 340 m above MSL. A random sampling was done only in the uncultivated wastelands since additional water and nutrients from cultivated fields adversely influence the nutritional composition of seeds. Each sampling included ~ 100 plants (per location) which were free from any pest/disease, herbivorous disturbance and human activities. Every sampling location included three sampling points in proximity, within ~ 100 m2. None of the collection sites was inside the national park or restricted forest area, so prior permission was not required for the collection of P. amarus. The collected specimens were soon taken to the lab in a zip-lock cover for observation and processing. The mature seeds were harvested from the randomly selected shade-dried plants. It was observed that the number of seeds in the field-collected plants varies from ~ 50 to 150.
2.2 Storage of seeds
The collected seeds were stored for a specific duration of time and they were checked periodically for its germinability. Initially, we separated the seeds that sink and float in water for its germination differences, with respect to imbibition time. The unsorted seeds were stored in four different storage conditions such as seeds with moisture in room temperature (set 1), without moisture (set 2) under room temperature, seeds with moisture at 4 °C (set 3) and seeds without moisture under 4 °C (set 4) under refrigeration. Moisture-free conditions were achieved by removing the excess moisture in the ambience by using self-indicating dry silica gel beads 100 g for desiccation  and for sustaining moisture, tissue paper soaked in distilled water in sealed plastic containers . The water-soaked tissue paper was replaced every 2 weeks, and silica beads were replaced when required (based on colour change). After every month, the germination percentage was checked for 100 seeds in triplicate from each set.
2.3 Moisture content
2.4 Morphological and molecular identification of P. amarus
2.5 Germination of P. amarus
For the germinability test, 1 g of visibly healthy seeds (i.e. free of microbial colonisation, decay and herbivore damage) were separated into sink and float seeds by a method proposed by Edson et al.  and assessed using a method described by Karthik et al. . A seed was considered as germinated when there was an emergence of the radicle, and the observation of seed germination was recorded daily.
2.6 Biochemical contents of the P. amarus seeds
Biochemical contents of both sink and float seeds were measured from 100 mg of seeds. The total protein, carbohydrate and lipid content of the seeds were estimated by following the method of Bradford , Dubois et al. , and Folch et al. , respectively.
2.7 Statistical analysis
All the laboratory experiments were carried out in completely randomized block design (CRBD), and each independent experiment was repeated at least three times with three replications for confirmation of the results. The effects of seed storage conditions were analysed by analysis of variance (ANOVA), and treatment means were compared by Duncan’s Multiple Range Test (DMRT) at p ≤ 0.05. The difference between the seed types (sinking and floating) was analysed by Chi square (χ2) tests and the treatment means were compared by DMRT at p ≤ 0.05. The germinability response of seed storage under different conditions and time was assessed by the best-fit (regression) equation based on the highest R2 value. All the graphs were constructed using SigmaPlot® (Ver. 10.0 for Windows), and statistical analysis was performed using RStudio© (Ver. 1.1.456 for windows).
3.1 Morphological and molecular identification of P. amarus
3.2 Germination of P. amarus seeds
Time period of shadow drying for collection of viable seeds
Collection of seeds (days)
58 ± 4.39a
29 ± 3.10a
27 ± 3.63b
13 ± 2.08b
15 ± 2.23c
6 ± 1.41c
6 ± 1.41d
2 ± 0.81d
3.3 Storage of P. amarus seeds
To protract germinability, the sink seeds harvested between 0 and 3 days were stored in different temperature and moisture conditions, and the germination percentage was checked after every month. The result shows that there is a significant variation among the seeds stored when subjected to different temperature and moisture (Fig. 4b). Seeds stored at room temperature both RT + M (room temperature with moisture) and RT reduced their viability drastically and completely lost their germinability within 4 months. However, seeds stored at 4 °C showed extended viability than their counterparts. Germinability was comparable up to five months with 4 °C + M stored seeds, nevertheless the seeds stored at 4 °C edge over later and have 2 months extended germinability up to 12 months.
3.4 Biochemical contents of the seeds
In trade, the medicinal herb Phyllanthus has a wide demand, but as a species, it is often wrongly identified and hence leads to unproductive benefit. Many of the Phyllanthus species belonging to the subsection ‘Swartziani’ grow in close proximity in India and their morphological identification is not easy. The confusion connected to the name P. niruri is due to Linnaeus’s inclusion of synonyms which actually belong to different species, namely P. amarus Schum. & Thonn., P. debilis Klein ex Wild., Phyllanthus madraspatensis L., P. urinaria. The leaf shape character of P. amarus is oblong with arounded tip, whereas in P. fraternus, the leaf tip is obtuse. The P. debilis leaf is narrow elliptical in the upper region and cuneate at the lower end and the leaf tip is acute. The P. maderaspatensis leaf is spathulate and its leaf tip is oblong-elliptic . The P. urinaria leaf is oblong, mucronate below leaves reddish-green seeds is seen .
To distinguish the Phyllanthus species, Jain et al.  developed SCAR markers for molecular characterisation of four species of the niruri complex (P. amarus, P. fraternus, P. debilis and P. urinaria) using MAP 09 and MAP10 primers. The MAP primers exhibited a distinct polymorphism that are species-specific DNA fragments, 1150 bp for P. amarus, 317 bp for P. fraternus, 980 bp for P. debilis and 550 bp for P. urinaria. The results obtained by them were in accordance with the result of the present study, with 1150 bp amplicon obtained with field-collected P. amarus (Fig. 2).
Based on the SEM seed topology study of Machado et al. , only P. amarus seed consists of six-to-nine regular longitudinal ribs with transversal finger-shaped rodlets than its counterparts Phyllanthus tenellus Roxb., P. niruri L., Phyllanthus stipulates (Raf.) Webster, P. urinaria L., Phyllanthus caroliniensis Walt. The present study using SEM also resulted in the distinguishable presence of six-to-seven regular longitudinal ribs and confirmed the collected seeds as P. amarus (0.8 mm long and 0.8–0.6 mm width) whereas P. debilis (Seeds 1 mm long and 0.6 mm wide, longitudinally 6–7 ribs and coloured pale yellowish-brown)  and P. madraspatensis are trigonous with 1.5 mm length and a shiny dark-brown colour .
The germinability of natural P. amarus seeds was low. Therefore, harvesting and storage at the right time is very essential to produce good germinable seeds. The present study demonstrated that the seeds harvested within 0–3 days were matured and separated them from the plants. These also had a higher sink percentage and good germinability. Based on the conceptual study of Unander et al. , the germination percentage and duration of shade drying of P. amarus plants could be related to the dehiscence colour of the seeds. All the seeds collected within the first 30 h were dark green in colour and had more sink seeds and better germination (Table 1). Seeds collected after 96 h are light tan in colour and have significantly fewer sink seeds and lower germination percentage. From the results, it was apparent that the collection of dark green sink seeds is necessary for successful germination. On the other hand, light tan float seeds result in poor germination and are to be avoided in ex situ conservation.
The results of seed storage conditions revealed that a warm and wet environment seriously affects the germinability of the P. amarus seeds. However, seeds stored at 4 °C can improve the viability (Fig. 4). Unander et al.  reported that P. amarus seeds have a longer viability of up to 18 months when stored at − 20 °C but the germination percentage declines over time. The results of the present study also report a similar trend: seeds stored at a lower temperature have maximum viability with a decreasing seed germination rate. Earlier studies have found that the germination percentage of older seeds increase because a slightly longer period of storage could eliminate germination inhibitors; therefore, they tend to germinate faster when compared to fresh seeds . Fresh seeds had seedlings within 13 days and then upon storage for 6 months at room temperature, the time required for the seedlings was drastically reduced to only 10 days. Although, the rate at which the germination takes place was quick and their germination success was dropped in all conditions.
From the earlier report of Pius et al. , the biochemical composition of P. amarus seeds very similar our sink seed composition (Fig. 5). The starch stored inside the seeds remains insoluble in water , where the prime enzyme cell wall-bound invertase breaks it down into glucose during germination and helps the mitotic cell division . Hence, the distribution and concentration of glucose are the main criteria which directly correlate to the maturity and gain of weight to the seeds that make them sink.
Due to the assimilation of stored lipids at the time of maturity, the concentration was low in the sink seeds which displayed a quick saturate in the water as they sink, whereas the float seeds remain immature and their initial stored lipids remain non-polar, thus helping them float. Moreover, the non-polar lipids prevent water into protoplasm and thus prevent germination. Our results in P. amarus sink seeds have less lipid content by 33% than that of the float. So, there might be difficult for the water to reach germplasm of the float seeds. Thereby, the germination of float seeds could not be accomplished . As a result of germination, it was understood that mature sink seeds break down their stored lipids for complete development. The lipids are highly non-polar hydrocarbons which are insoluble in water as compared to polar substances like glucose, which solubilise with a saturation point of 1.1 mg/g of water. Due to the presence of higher lipid, it makes them float; further, it blocks the entry of water into the germplasm through the testa . A significantly higher percentage of germination can be related to the presence of a higher total protein content in the sink seeds (Fig. 5) due to the presence of many active enzymes compared to float seeds which has lower level of protein in this species .
Habitat destruction of the medicinal plant P. amarus has encounterd a serious threat due to urbanisation, climatic changes and their raw demand. As described in this study, P. amarus has a low germination rate and its germination rate drastically reduces with time. In order to conserve the seeds, both the selection of viable seeds and condition of storage are major factors in the longevity of the seeds. It was observed that sink seeds have the ability to germinate but not float seeds. Sink seeds ratio was high during the first 3 days of shade drying. Storage at 4 °C without moisture resulted in seed viability for up to 12 months. So, this information provides a suitable parameter and criteria for P. amarus germination.
The authors are grateful to farmland owners for permitting us to collect the sample. We also thank UGC-CPEPA, New Delhi, India, for providing financial support (B-2/2008; NS/PE) to carry out this work. We thank the Director, Centre for Advance Studies in Botany, University of Madras for providing the scanning electron microscope.
Compliance with ethical standards
Conflict of interest
The authors declare that they have no conflict of interest.
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