Botanica https://doi.org/10.35513/Botlit.2022.1.9 2022, 28(1): 75–80 ISSN 2538-8657 Original research Reproductive biology of green-stemmed and red-stemmed Basella alba Abolade Oluremi Bolaji 1*, Atanda Samuel Oladejo 2, Oluwatobi Isaac Adeniran 1 1 Obafemi Awolowo University, Department of Botany, Ile-Ife, Nigeria Obafemi Awolowo University, Department of Crop Production and Protection, Ile-Ife, Nigeria *Corresponding author. E-mail: abolaji@oauife.edu.ng 2 Abstract Bolaji A.O., Oladejo A.S., Adeniran O.I., 2022: Reproductive biology of green-stemmed and red-stemmed Basella alba. – Botanica, 28(1): 75–80. https://doi.org/10.35513/Botlit.2022.1.9 Basella alba is an important, underutilised vegetable rich in vitamins and consumed in many parts of the world. However, the reproductive biology of this species is insufficiently known; therefore, this study was performed aiming to fill this knowledge gap. Although green-stemmed and red-stemmed Basella alba share a lot of reproductive attributes in common, the obtained results showed differences in their flower bud apex colour, anther colour, spike colour, days to 50% flowering, plant height at flower bud initiation and the mean number of flowers per spike. In both forms, the floral attributes favoured self-pollination. However, the possibility of crosspollination among them due to the activities of pollinators such as ants, moths, butterflies and bees suggests a mixed mating system. Furthermore, both forms attained sexual maturity at varying periods, thus indicating the existence of a temporal prezygotic barrier between them and limiting the chances of their hybridisation. They could also be responsible for the ability to remain in their distinct forms even when they exist together in the same habitat. Keywords: germination percentage, isolating mechanism, pollen viability, self-pollination. INTRODUCTION The genus Basella (Basellaceae) comprises five species: Basella excavata Eliot, Basella leandriana H. Perrier, Basella madagascariensis Boivin ex H.Perrier, Basella paniculata Volkens and Basella alba L. (Eriksson, 2004). Basella excavata, Basella leandriana and Basella madagascariensis are native to Magadascar, Basella paniculata is native to South and East Africa, while Basella alba has a pantropical distribution (Eriksson, 2004). Earlier recognised Basella rubra is currently considered a red-stemmed form of Basella alba (Cook, 2010; Deshmukh & Gaikward, 2014). Green-stemmed and red-stemmed forms of Basella alba are common vegetables among the Yoruba tribe of Southwestern Nigeria and are referred to as ‘amunututu’. They are good sources of vitamins A and C (Roy et al., 2010). They contain several phytoconstituents such as proteins, alkaloids, carbohydrates, polysaccharides, phenols, flavonoids, carotenoids, minerals and vitamins (Kumar et al., 2012). They are helpful in phytomedicine in treating gonorrhoea, constipation, leprosy, dysentery, ulcer and burns (Dixit & Goyal, 2011; Shantha et al., 2016). Although there have been reports on some aspects of the morphology and phytomedicinal potentials of green-stemmed and red-stemmed Basella alba, there 75 Bolaji et al. || Reproductive biology of Basella alba is a lack of information on their reproductive biology. The specific objectives of this research were, therefore, to investigate their reproductive attributes such as seed germination percentage, days to seedling emergence, floral structure, period of anthesis, pollen viability, days to 50% flowering, plant height at flower bud initiation, pollinators, mode of fruit and seed dispersal and reproductive isolating mechanisms. MATERIALS AND METHODS Seeds of green-stemmed and red-stemmed Basella alba accessions were collected from various locations within Southwestern Nigeria (Table 1). These were authenticated at the IFE Herbarium located at the Department of Botany, Obafemi Awolowo University, Nigeria. Seeds were sown in polythene bags filled with sterilised topsoil and raised to maturity in the screen house of the Department of Botany, Obafemi Awolowo University, Ile-Ife, Nigeria. The experiment was laid out in an utterly randomised design while regular agronomic practices such as irrigation, weeding and staking for optimal growth and devel- opment were carried out regularly. A total number of 38 seedlings of green-stemmed Basella alba accessions and 45 seedlings of red-stemmed Basella alba accessions were raised and studied. The study was carried out from December 2018 to April 2020. Data were collected from randomly selected plants on the following parameters: the mean number of days to seeding emergence, mean germination percentage, the mean number of days to 50% flowering, floral attributes, mean plant height at flower bud initiation, period to anthesis, percentage pollen viability, types of pollinators, the mean number of fruits per spike, mean number of seeds per spike, the colour of ripe and unripe fruit, colour and types of seeds and mode of seed dispersal. The germination study was carried out on 8 cm Petri dishes laid with 7 cm Whatman filter paper at temperatures between 20°C and 30°C. Twenty seeds drawn at random were placed on a wet filter paper inside a Petri dish, and this was done in three replicates. The mean number of days to the radicle emergence was documented, and the percentage of germinated seeds was determined. Table 1. Sources of green-stemmed and red-stemmed Basella alba plants used in the study. Numbers in brackets represent the number of studied plants within the accession Accession BAIWO (10) Form Green-stemmed Iwo BAIFE (8) Green-stemmed Ile-Ife BAONDO (5) Green-stemmed Ondo BAEKITI (5) Green-stemmed Ekiti BAOYO (5) Green-stemmed Oyo BALAG (5) Green-stemmed Ijede BRIWO (9) Red-stemmed Iwo BRIFE (8) Red-stemmed Ile-Ife BRONDO (10) Red-stemmed Ondo BREKITI (7) Red-stemmed Ekiti BROYO (5) Red-stemmed Ogbomoso BRLAG (6) Red-stemmed Ijede 76 Source Location 7.629444 °N 4.191111 °E 7.523056 °N 4.515833 °E 7.236111 °N 5.239722 °E 7.616389 °N 5.218333 °E 7.419167 °N 3.964722 °E 6.942778 °N 4.191111 °E 7.629444 °N 4.191111 °E 7.523056 °N 4.515833 °E 7.236111 °N 5.239722 °E 7.616389 °N 5.218333 °E 8.146111 °N 4.259167 °E 6.942728 °N 3.098056 °E Comment Cultivated in home gardens Cultivated in school gardens Cultivated in home gardens Cultivated in gardens Cultivated in church gardens Cultivated beside home Cultivated in home gardens Cultivated in school gardens Cultivated in home gardens Cultivated in home gardens Cultivated in school gardens Cultivated in home gardens Bolaji et al. || Reproductive biology of Basella alba The pollen viability study was carried out by collecting pollen grains from freshly dehisced anthers onto microscope slides. These were stained with cotton-blue-in-lactophenol for 30 minutes. A total of 100 pollen grains were examined using the light microscope for percentage stainability following the methods of Bolaji & Nwokeocha (2013). The wellformed and deeply stained pollens were considered viable, while those with a collapsed outline, partially stained or not stained, were considered non-viable. The quantitative reproductive data obtained were subjected to the General Linear Model (GLM) analysis of variance (ANOVA). Results of data analysis were presented as the mean and standard error (mean ± SE). In addition, differences between means were evaluated by applying Duncan Multiple Range Test (DMRT) at p < 0.05. Statistical analyses were performed employing System Analysis Software (SAS, version 9.0). Table 2. Comparison of reproductive characteristics between green-stemmed and red-stemmed forms of Basella alba. Different letters in superscripts across a row indicate significant differences between the means (p < 0.05) Attributes Green-stemmed Red-stemmed (n = 38) (n = 45) Percentage of seed germination (%) 72.40 ± 0.97a 75.50 ± 0.48a Number of days to seedling emergence 10.12 ± 3.15a 13.02 ± 4.11a Plant height at flower bud initiation (cm) 87.70 ± 9.05a 119.49 ± 6.80b Days to 50% flowering 100.10 ± 3.60a 200.20 ± 4.07b Mean number of flowers per spike 22.16 ± 3.13 16.45 ± 1.83b 93.70a 94.50a Mean fruit length (cm) 1.07 ± 0.03a 1.15 ± 0.02a Mean fruit diameter (cm) 2.20 ± 0.02a 2.25 ± 0.03a RESULTS Mean number of fruits per spike 12.21 ± 4.59a 10.24 ± 3.20a Analysis revealed that germination of greenstemmed Basella alba seeds was 72.40 ± 0.97%, while that of red-stemmed Basella alba was 75.5 ± 0.48%, and no significant differences between them were found (Table 2). The mean number of days to seedling emergence was 10.12 ± 3.15 for greenstemmed and 13.02 ± 4.11 for red-stemmed plants. The mean plant height at flower bud initiation was 87.70 ± 9.05 cm for green-stemmed Basella alba, while 119.49 ± 6.80 cm for red-stemmed plants. Significant differences were found between plant height at flower bud initiation (p = 0.005). The mean number of days to 50% flowering was 100.10 ± 3.60 for green-stemmed, whereas red-stemmed plants spent significantly longer (p < 0.001) time to flowering, i.e. 200.20 ± 4.07 days (Table 2). The study revealed that the mean number of flowers per spike of green-stemmed plants was significantly higher (p < 0.001) than of red-stemmed plants (22.16 ± 3.13 and 16.45 ± 1.83 flowers, respectively). Anthesis of both forms of Basella alba occurred between 8:00 a.m. and 12:00 noon. The pollens of green-stemmed plants were powdery, light yellow. The pollens of red-stemmed Basella alba differed by deep yellow colour. No significant differences between the mean pollen viability were found (93.70% and 94.50 %, respectively). Mean seed length (cm) 0.65 ± 0.01a 0.64 ± 0.04a Mean seed diameter (cm) 1.25 ± 0.04a 1.20 ± 0.02a Percentage pollen viability (%) a The fruits of Basella alba are spherical juicy drupes. The unripe fruits were green and deep purple when they ripened for both forms of this species. The mean fruit length was 1.07 ± 0.03 cm and mean diameter was 2.20 ± 0.02 cm for green-stemmed Basella alba, while the mean fruit length was 1.15 ± 0.02 cm and mean diameter was 2.25 ± 0.03 cm for red-stemmed Basella alba. No significant differences between the two forms of Basella alba by the mean fruit length (p = 0.638) and fruit diameter (p = 0.223) were revealed. The mean number of fruits, and thus seeds, per spike was 12.21 ± 4.59 for green-stemmed Basella alba, while red-stemmed plants had 10.24 ± 3.20 (Table 2). According to the mean number of fruits per spike, no significant differences were found between the two forms (p = 0.407). The mean seed length was 0.65 ± 0.01 cm, while the mean diameter was 1.25 ± 0.04 cm for greenstemmed Basella alba. The mean seed length of red-stemmed plants was 0.64 ± 0.04 cm, and their mean diameter was 1.20 ± 0.02 cm (Table 2). No significant differences were found between the green77 Bolaji et al. || Reproductive biology of Basella alba stemmed and red-stemmed plants’ mean seed length (p = 0.352) and seed diameter (p = 0.374). The two forms flowered and fruited sequentially throughout the remaining period of their life cycles. DISCUSSION Reproductive biology and systematics are interrelated (Anderson et al., 2002). Its importance in the taxonomic description of species has been harnessed by many researchers (Folorunsho & Olorode, 2008; Ratha & Paramathma, 2009; Bolaji et al., 2020). This study revealed that green-stemmed and red-stemmed Basella alba plants were similar in their reproductive characteristics. There were no significant differences between the two Basella forms regarding seed germination percentage and the number of days to seedling emergence, pollen viability, mean fruit length, mean fruit diameter, the mean number of fruits per spike, mean seed length and mean seed diameter (Table 2). The lack of significant differences in these traits supports the opinion that the two Basella forms belong to the same species. This point of view has been expressed by Roy et al. (2010). Although the inflorescence, fruits and seeds were similar in many respects, notable differences were observed in their flower bud apex and anther colours. While the flower bud was pink and the anther light yellow in green-stemmed Basella alba, the flower bud apex was deep purple, and the anthers were deep yellow of red-stemmed Basella alba. This could be why some researchers (Henry et al., 1987; Roy et al., 2010) consider them as varieties. Understanding species’ reproductive biology helps clarify characters’ potential use and values in systematic treatments (Anderson et al., 2002). The percentage germination of the seeds was notably high (72.40 ± 0.97 for green-stemmed and 75.50 ± 0.48 for red-stemmed Basella alba). According to Labhane et al. (2014), the traditional method of studying the viability of seeds is by considering the percentage of their germination. The number of days to seedling emergence for both forms was similar (10.12 ± 3.15 for green-stemmed and 13 ± 4.11 for red-stemmed Basella alba). The high viability of the seeds and the ability of the seedlings to emerge within a few days could have significantly contributed to their reproductive success and ability to thrive easily 78 under varying environmental conditions. According to Norman et al. (2021), seed germination and seedling emergence are the most critical and vulnerable phases of a crop cycle. Therefore, they can predict the extent of a speciesʼ success. Palada & Crossman (1999) have also noted that Basella alba adapts easily to various soils and climates and is considered one of the best tropical leaf-vegetable throughout the tropical world. It is noteworthy that from the number of days to 50% flowering and plant height at flower bud initiation, the red-stemmed Basella rubra spent a much longer time (Table 2) in the vegetative phase than the green-stemmed. This implies that the two Basella alba forms attain sexual maturity at varying periods. Conversely, even though the period of anthesis, pollinators, fruit and seed dispersal agents were similar for both Basella alba forms, the timing of their sexual maturity is such that green-stemmed Basella alba would have flowered and started to produce fruits long before red-stemmed Basella alba begins to flower, thereby making hybridisation between the two forms limited. This indicates the temporal prezygotic barrier, which could be responsible for their ability to remain in their distinct forms even when they occur in the same habitat. Prezygotic barriers include spatial, temporal or behavioural differences leading to sexual isolation, and they are the most critical and effective barriers, given that they act early in the life cycle of an organism to impose the strongest impediment to gene flow, thereby preventing hybridisation (Widmer et al., 2009; Henrich & Kalbe, 2016). The bisexual nature of the flowers and the location of the anthers above and very close to the stigma, favour self-pollination in both forms. However, the bright colouration of the perianths of the two species attracted pollinating insects such as ants, bees, moths and butterflies, thereby encouraging crosspollination, suggesting that the breeding system in both forms is a mixed mating system. According to Holsinger (1996), the continuum between self-pollination and outcrossing in plants results in a mixed mating system. Understanding the breeding system of these two Basella alba forms could enhance their effective genetic improvement through hybridisation programmes by breeders, thereby enhancing their utilisation by consumers. It is also key to their ef- Bolaji et al. || Reproductive biology of Basella alba fective conservation. According to Anderson et al. (2001), the knowledge of the reproductive system of plants is a central element in their effective conservation. Vivian-Smith et al. (2007) have also noted that flowers of Anredera cordifolia (Basellaceae) attract pollinators such as bees and ants. Hybridisation between green-stemmed and redstemmed Basella alba could be enhanced by varying their planting periods. Their flowering periods overlap so that outcrossing between them is enhanced. This is worthwhile considering the temporary prezygotic isolation between them because of differences in the time it takes for both species to attain the generative stage. Breeders could explore this information in carrying out hybridisation programmes for the genetic improvement of Basella alba. The knowledge of pollination, breeding systems and seed dispersal of plant species could be used to enhance their conservation and restoration (Hamrick et al., 1991; Karron, 1991; Ramirez, 2006). We suppose that humans mainly disperse seeds of both forms of Basella alba. Vivian-Smith et al. (2007) have reported that Anredera cordifolia (Basellaceae) is also primarily spread by humans and water. ACKNOWLEDGEMENTS The authors gratefully thank the laboratory staff members of the Department of Botany, Obafemi Awolowo University, Ile-Ife, Nigeria, for providing some of the facilities used in this work. 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