Morphological characterization and cytological studies of the green-stemmed and the red-stemmed Basella alba

Basella alba is an underutilized vegetable with ethnobotanical importance used for culinary as well as medicinal purposes in many parts of the world. Morphological characterization and chromosome studies of the mitotic and meiotic cells of the green stemmed and the red-stemmed B. alba was carried out with a view to filling the knowledge gaps that exist in their morphological characterization and also to provide insightful information on their chromosome numbers and meiotic behaviour. The B. alba accessions studied were characterized with respect to their habit, leaves, inflorescence, fruits and seeds. Mitotic and meiotic studies were carried out on the Basella accessions using standard techniques. The morphological studies revealed significant differences between the green-stemmed and red-stemmed Basella alba with respect to the green/red colour of their stems, colour of the flower bud apex, mean plant height at flower bud initiation, mean leaf length, mean leaf width, mean petiole length, mean flower per spike, mean fruit length and mean fruit diameter. The cytological study revealed a chromosome number of 2n=4x=44 for both the green-stemmed and the redstemmed B. alba studied. It also revealed the occurrence of chromosomal aberrations such as stickiness and precocious migration of chromosomes to the poles during meiosis, which could lead to irregular chromosome segregation that could result in chromosome instability and aberrant meiotic products within the species.


Introduction Introduction Introduction Introduction
Basella alba L. is the most common species in the family Basellaceae (Ozela et al., 2007) and it comprises the green-stemmed (Basella alba var. alba) and purple-stemmed (Basella alba var. rubra) Basella alba forms. It is native to tropical Southern Asia and probably originated from India or Indonesia (Saroj et al., 2012). B. alba is particularly abundant in Malaysia, Philippines, Thailand, Nepal, India, tropical Africa, the Caribbean and tropical South America (Palada and Crossman, 1999).
Basella alba is an underutilized vegetable often cultivated in home gardens (Bolaji et al., 2022a) and used for culinary and medicinal purposes. It is a very popular vegetable in many communities of South-western Nigeria and is also one of the chief sources of the major ingredients in the Northern Nigeria and North-eastern Nigerian foods (Izonfuo et al., 2006). It is useful as medicinal plant in the treatment of various ailments including anaemia (Rahmatullah et al., 2010), pelvic inflammation disease, threatened abortion (Focho et al., 2009), hypertension (Olowokudejo et al., 2008), earache and sore throat (Chatchawal et al., 2010;Paul et al., 2011), gonorrhoea (Singh et al., 2010), diabetes (Shantha et al., 2016) and ulcer (Dixit and Goyal, 2011).
Chromosomes are key building blocks of eukaryotic genomes (Tiang et al., 2012) and the cytogenetic characterisation of species require in-depth studies of the chromosome numbers and chromosomal behaviours of the species and their morphogenic varieties (Silva et al., 2017). Meiosis, a process by which sexually reproducing organisms reduce their genome from diploid to haploid (John, 1990) is a highly coherent and genetically controlled process (Pessim et al., 2015) which involves the process of commitment and initiation, homologous chromosome pairing, synapsis, inter-homologous reciprocal recombination, disjunctive segregation and haploid gamete formation (Murphy and Bass, 2012). There were knowledge gaps concerning the morphological characteristics of the green-stemmed and the red-stemmed Basella alba. Previous reports had focused on their stem colour, leaf shapes, floral arrangement and fruit shape (Palada and Chang, 2003;Adeyemi, 2007;Kumar et al., 2013). Previous cytological studies on the genus Basella indicated varying reports on the chromosome numbers of the green-stemmed and the red-stemmed B. alba., with the reports varying from chromosome numbers 2n = 44 to 2n = 45 and 2n = 48 (Hanson et al., 2005;Silva et al., 2017). There is no known report of chromosome numbers of B. alba morphotypes from Nigeria. There is also paucity of information about the meiotic behaviour of B. alba. The specific objectives of these study were therefore to characterize the green-stemmed and red-stemmed B. alba and establish their chromosome numbers as well as investigate their meiotic behaviours.

Plant source and identification
The seeds of the Basella alba accessions studied were collected from various locations in Nigeria (Table  1) and authenticated at the IFE Herbarium, Department of Botany, Obafemi Awolowo University, Nigeria. The seeds were sown in polythene bags filled with sterilized top soil laid out in a completely randomized design and raised to maturity in the screen house of the Department of Botany, Obafemi Awolowo University Ile-Ife, Nigeria. Normal agronomic practices such as watering, weeding and staking were performed.

Morphological characterization of plants studied
The qualitative and quantitative attributes of the green-stemmed and the red-stemmed Basella alba accessions studied were characterized with respect to their habit, leaves, inflorescence, fruits and seeds. The quantitative data were collected in five replicates for thirty-eight (38) accessions for both the green-stemmed and the red-stemmed. The quantitative morphological attributes of the green-stemmed and red-stemmed B. alba were compared by subjecting the data obtained to the Generalized Linear Model (GLM) analysis of variance (ANOVA) and the differences between the means that were significantly different at p<0.05 were evaluated by Duncan Multiple Range Test (DMRT) using System Analysis Software (SAS) version 9.0. The means were presented as mean and standard error (mean ± SE).

Pollen viability and seed set study
The pollen viability study was carried out on 100 pollen grains following the method of Bolaji et al. (2022b). Pollen grains from freshly dehisced anthers were harvested on to microscope slides and were stained with cotton-blue-in-lacto-phenol for 30 minutes. The well-formed and deeply stained pollens were considered as viable; while those with collapsed outline, partially stained or not stained were considered to be non-viable. The percentage of viable pollens were documented.
The seed set study was carried out on 10 randomly selected plant. The percentage seed set was determined by dividing the number of seeds obtained by the number of seeds expected (i.e., number of flowers obtained) multiplied by 100, following the method of Idowu and Oziegbe (2017).

Mitotic chromosome studies
Young whole plants of the green-stemmed and the red-stemmed B. alba were placed in plastic bottles containing water and allowed to root. The root tips were excised and fixed in 1: 3 acetic-ethanol after four days between 9:00 a.m. and 11:00 a.m. The fixed root tips were left at room temperature for 48 hours before being used for preparation of mitotic cells. The root tips were hydrolysed in 18% HCl, squashed and stained with modified Orcein for 15 minutes following the methods of Bolaji et al. (2019). Slides prepared were observed under the light microscope and chromosome counts were made for each of the Basella forms. Good mitotic spreads were photomicrographed using Amscope MT Microscope camera version 3.0.0.1 attached to light microscope.

Meiotic chromosome studies
Young flower buds were harvested into vias containing 1: 3 acetic-ethanol between 9:00 a.m and 11:00 am. The anthers were excised, squashed and stained in FLP orcein (2g of Orcein in 100 cm 3 of solution containing equal parts of formic acid, lactic acid, propionic acid and distilled water). Pollen mother cells were examined to investigate meiotic events and photomicrographs of good meiotic spreads were taken using Amscope MT microscope camera version 3.0.0.1 attached to a light microscope.

Description of the green-stemmed and red-stemmed Basella alba studied
The green-stemmed ( Figure 1A) and red-stemmed ( Figure 1B) Basella alba studied were perennial, herbaceous twinning, with glabrous stem ( Table 2). The mean plant height was 87.65 ± 5.32 cm for the greenstemmed, while it was 119.49 ± 6.99 cm for the red-stemmed. The red-stemmed had significantly greater plant height (Table 3) than the green-stemmed at P = 0.0005. The mean stem diameter for both forms of B. alba were not significantly different. The green-stemmed had a mean stem diameter of 3.38 ± 0.09 cm, while the red-stemmed had 3.64 ± 0.15 cm. The mean number of primary branches was 5.26 ± 0.45 for the greenstemmed and 4.34 ± 0.29 for the red-stemmed with no significant difference between them. The mean internode distance was 4.58 ± 0.23 cm for the green-stemmed and 5.05 ± 0.31 cm for the red-stemmed., with no significant difference between them. The leaves were green, simple, glabrous, succulent, cordate, margin entire, apex acute and round, venation pinnate, leaf arrangement opposite at seedling stage, alternate at mature stage, leaf attachment petiolate; leaves exstipulate for both forms of B. alba ( Table 2). The mean leaf length was 6.62 ± 0.37 cm for the green-stemmed and 7.75 ± 0.31 cm for the red stemmed, with the red-stemmed having significantly longer leaves than the green-stemmed at P = 0.0218 (Table 3). The mean leaf width was 5.23 ± 0.19 cm for the greenstemmed and 6.67 ± 0.26 for the red-stemmed, with the red-stemmed having significantly wider leaves at P = 0.0001. The mean petiole length was 1.71 ± 0.06 cm for the green-stemmed and 1.95 ± 0.08 cm for the redstemmed with the red-stemmed having significantly longer petiole than the green-stemmed at P = 0.0172. The inflorescence was racemose with each of the flowers consisting of one perianth with 5 segments joined at the base for both forms of B. alba ( Table 2). The perianth was cream with pink apex ( Figure 1C) in the green stemmed, while it was cream with deep purple apex ( Figure 1D) for the red-stemmed. Bracts were attached to the base of the perianth in both forms. The flowers were bisexual, pedicel sessile and glabrous, symmetry radial; anther 5, pollen yellow, powdery; filament white with adnate attachment; stigma simple, glabrous; style of unequal length, white, simple, glabrous; ovary superior, placentation basal for both forms of B. alba studied. The mean flower per spike was 22.16 ± 0.93 for the green-stemmed while it was 16.45 ± 0.82 for the red stemmed, with the green stemmed having significantly higher number of flowers per spike than the red-stemmed at P = 0.0001 (Table 3). The fruits were berry, glabrous, spherical, apedicelate on green spike; four lobed, one-seeded, unripe colour green, ripen colour deep purple ( Figure 1E and Figure 1F) for both forms of B. alba studied. The mean fruit length was 1.07 ± 0.03 cm for the green stemmed while it was 1.15 ± 0.cm for the red stemmed, with the red-stemmed having significantly longer fruits than the green-stemmed at P = 0.0398 (Table 3). The mean fruit diameter was 2.12 ± 0.02 cm for the green-stemmed while it was 2.25 ± 0.03 cm for the red-stemmed with the red-stemmed having significantly wider fruits than the green-stemmed at P = 0.0002. The mean number of fruits per spike was 12.21 ± 0.69 for the green-stemmed, while it was 10.24 ± 0.74 for the red-stemmed with no significant difference between them.
The seeds were brown ( Figure 1G and Figure 1H), spherical in shape for both forms of B. alba studied. The mean seed length was 0.66 ± 0.10 cm for the green-stemmed, while it was 0.64 ± 0.02 cm for the redstemmed with no significant difference between them (Table 3). The mean seed diameter was 1.25 ± 0.01 cm for the green-stemmed, while it was 1.26 ± 0.01 cm for the red-stemmed with no significant difference between them. The mean number of seed per spike was 12.21 ± 0.69 for the green-stemmed while it was 10.24 ± 0.74 for the red-stemmed with no significant difference between them.

Chromosome studies
The study revealed a diploid chromosome number of 2n = 44 and haploid chromosome number of n = 22 for the green-stemmed (Figure 2A, Figure 2B, Figure 2C) and the red-stemmed Basella alba ( Figure 2D) studied. The meiotic chromosome study also revealed the occurrence of many quadrivalent chromosomes amidst bivalent chromosomes at diakinesis ( Figure 2C) and metaphase I ( Figure 2D). It also revealed the occurrence of chromosome stickiness ( Figure 2E) and precocious migration of chromosomes to the poles ( Figure 2E). The frequency of the chromosome stickiness was 2.5% in the green-stemmed B. alba; while it was 3.5% in the red-stemmed (Table 4). The frequency of the precocious migration of chromosomes to the poles was 5.7% in the green-stemmed; while it was 6.3% in the red-stemmed.  The green-stemmed and red-stemmed Basella alba studied were similar with respect to many of their vegetative and reproductive attributes (Table 3) and this is in support of their taxonomic classification as members belonging to same species as reported by Roy et al. (2010), Bolaji et al. (2022a), andBolaji et al. (2022b). However, there were significant differences between them with respect to the green/red colour of their stems, colour of the flower bud apex (i.e., colour of the perianth apex), mean plant height at flower bud initiation, mean leaf length, mean leaf width, mean petiole length, mean flower per spike, mean fruit length and mean fruit diameter. These significant differences further support their delimitation as varieties belonging to same species. This is corroborated by the reports of Henry et al. (1987), Roy et al. (2010) and Bolaji et al. (2022a).
Chromosome counting from the floral and vegetative tissues of plants remains the most reliable method to establish their chromosome numbers (Mayrose et al., 2020). In this study, the diploid chromosome count of 8 2n = 44 and haploid chromosome count of n = 22 indicates a basic chromosome number of x = 11 thereby indicating a chromosome number of 2n = 4x = 44 for the green-stemmed and the red-stemmed Basella alba studied. This is in line with the reports of Silva et al. (2017) for Basella alba species obtained from Brazil. The chromosome numbers obtained for the green-stemmed and red-stemmed Basella alba in this study contradicts those of Hanson et al. (2005) who reported chromosome numbers 2n = 48, 41 and 44 for those obtained from England. It also contradicts the reports of Sperling and Bittrich (1993) who reported the basic chromosome number of x = 12 for B. alba. According to Silva et al. (2017) the chromosome number 2n = 44 observed in B. alba may have been derived from descending disploidy from the number 2n = 4x = 48.
The occurrence of many quadrivalent chromosomes amidst bivalent chromosomes at diakinesis and metaphase I further indicates that the green-stemmed and red-stemmed Basella alba studied were tetraploids. The precocious migration of some of the chromosomes to the pole and stickiness of the chromosomes during meiosis could lead to irregular chromosome segregation and chromosome instability within the species. Mendes-Bonato et al. (2001) noted that though the cause and biochemical basis for chromosome stickiness is not known, it is characterised by intense clustering and in severe cases makes it impossible for the chromosomes to separate leading to formation of single or varying numbers of pycnotic nuclei that culminate into full chromatin degeneration. Potapova and Gorbsky (2017) also noted that the full consequences of chromosome segregation errors are vast in scope and could produce aneuploid or polyploid cells as well as progeny with altered chromosome content. It is however noteworthy that in this study the frequencies of the chromosome stickiness and precocious migration of chromosomes to the pole during meiosis was very low (Table 4), hence could have been the reason why the pollen viability was still high and the percentage seed set was as well moderately high.

Conclusions Conclusions Conclusions Conclusions
This study characterized the accessions of the green-stemmed and red-stemmed Basella alba and established their chromosome as 2n = 4x = 44. The study also revealed significant differences between the green-stemmed and red-stemmed B. alba with respect to the green/red colour of their stems, colour of the flower bud apex, mean plant height at flower bud initiation, mean leaf length, mean leaf width, mean petiole length, mean flower per spike, mean fruit length and mean fruit diameter. The occurrence of many quadrivalent chromosomes amidst bivalent chromosomes suggest that they are tetraploids. The stickiness of the chromosomes and precocious migration to the poles during meiosis in the B. alba accessions studied could result in irregular segregation, which could possibly lead to chromosomal instability within the species. However, the frequencies of this aberrant chromosomal events were quite low in the B. alba accessions studied, hence, did not result in low pollen viability and low seed set.
All authors read and approved the final manuscript.

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Ethical approval Ethical approval Ethical approval Ethical approval (for researches involving animals or humans) Not applicable.