Karyotype Analysis and Ploidy Determination Using Flow Cytometry in African Bitter Milk Plant “Utazi”, Gongronema Latifolium Benth

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Karyotype Analysis and Ploidy Determination Using Flow Cytometry in African Bitter Milk Plant “Utazi”, Gongronema latifolium Benth

Peter O. Aikpokpodion*, Peggy A. Otu and Edak A. Uyoh

Department of Genetics and Biotechnology, Faculty of Science, University of Calabar, P.M.B 1115, Calabar, Nigeria

Received June 1, 2011; accepted November 28, 2011

Summary Availability of basic genetic information on a plant species is the first pre-requisite in designing any genetic improvement programme for the species. This study was therefore carried out to determine the karyotype of Gongronema latifolium to obtain basic knowledge on ploidy level and karyotype as a first step in understanding the genetics of this spice plant. Karyotype and flow cytometry (FCM) studies were carried out on 4 accessions of G. latifolium collected from the humid forest vegetation in Abia, Akwa Ibom, Cross River and Imo States of Nige ria. Chromosome number of Abia accessions were 2n=20 and 22 with karyotype formulae, 3m+4sm+2a+1t and 2m+4sm+ 2a+3t, respectively; Akwa Ibom accessions, 2n=18 with formula: 3m+5sm+1a; Cross River accessions, 2n=18 and 22 with formulae: 2m+4sm+2a+1t and 3m+4sm+3a+1t, respectively; Imo accessions, 2x=22 and 24 with formulae: 1m+5sm+3a+2t and 2m+7sm+2a+1t, respectively. Results obtained revealed cytotype variation and the occurrence of 4 cytotypes, 2n=18, 20, 22 and 24. We therefore conclude that G. latifolium is a diploid species with variable basic numbers. Four basic numbers, n=9, 10, 11 and 12, were therefore proposed for G. latifolium. FCM showed the presence of 2C and 4C DNA content and provided evidence for endoreduplication, a likely evolutionary survival mechanism in this climbing vegetable plant species. This is the first report of karyotype analysis in Gongronema latifolium, an important indigenous African vegetable spice plant with huge medicinal value.

Key words Karyotype, Gongronema latifolium, Endoreduplication, Flow cytomery method, Spice plant, ‘Utazi’.

Gongronema latifolium Benth Hook is an important medicinal plant of the family Asclepiadaceae with over 2000 species and some 250 genera widespread in tropical and subtropical regions especially in Africa, southern South America, northern and south eastern Asia with 44 genera and 270 species in China (Li et al. 2010). Some well-known tropical ornamental plants such as Orleander, Frangipani, Allamanda, Mandevilla also belong to this family. The sap of most plants in this family is a milky latex containing various alkaloids and glycosides, many of which are used in medicine and as insecticides (Karasov 2001). G. latifolium belongs to a genus of climbers and shrubs of deciduous and secondary forests known to have originated from west Africa and widely distributed in Nigeria, Ghana, Sierra Leone, Guinea Bissau, West Cameroon and other parts of Africa (Burkhill 1985, Okafor 2005, Nwinyi et al. 2008). It is widely dispersed in African forests and farms as wild, semi-wild and cultivated varieties (Odukoya et al. 2007). The leaves are simple and opposite, green to pale green measuring up to 4–8 cm long. Flowers are yellow and dioecious and actinomorphic while follicles are oblong-lanceolate with numerous seeds (about 50–60) strongly compressed with a coma. Ovary is usually superior and fruit is a

* Corresponding author, e-mail: [email protected] 44 P. O. Aikpokpodion et al. Cytologia 77(1) drupe, a berry, a capsule or a follicle (Walters and Keil 1996). The stem is soft and pliable and yields characteristic milky exudates when cut (GBIF 2007, Bullock 1961). Gongronema latifolium is popularly identified as ‘Utasi’ by the Efiks, Ibibios and Quas; ‘Utasi’ by the Igbos and ‘Arokeke’ by the Yorubas in Nigeria. It is a highly cherished spice and the parts eaten are the leaves and vines. It imparts a sharp bitter taste and sweet aroma to food, and increases appetite (Adelaja and Fasidi 2009, FAO 1983). The stems are used as chew sticks in Sierra Leone. Extracts or cold infu- sion of pounded leafy branches with lime juice expels intestinal worms, dispels stomach upsets and pains and tones the blood (Agbo et al. 2005). The use of crude leaf extract of this spice in maintain- ing healthy blood glucose levels has been reported (Okafor et al. 1994). Scientific studies have es- tablished the hypoglycemic, hypolipidemic and antioxidant properties of aqueous and ethanolic extracts of G. latifolium leaves (Ugochukwu et al. 2003, Ogundipe et al. 2003). Reports by various authors showed that G. latifolium contains essential oils, saponins and pregnanes among others and has anti-inflammatory properties (Schneider et al. 1993, Morebise and Fafunso 1998, Morebise et al. 2002, Eleyinmi et al. 2008). Abbiw (1990) records that the leaf serves as a vegetable used to prepare ‘pepper soup’ and is eaten raw in any quantity to check excesses of diabetes and hyperten- sion and to treat malaria and typhoid fever. Although of huge medicinal value with great potential for use in pharmaceutical industry, no basic genetic information on G. latifolium has been reported until now. This study was therefore conducted to determine the ploidy level and karyotype of Gongronema latifolium. This information will be invaluable for any subsequent genetic improvement of the crop. Karyotypes describe the number of chromosomes, and what they look like under a light microscope (King et al. 2006). Karyotype studies have been successfully conducted in Dianthus spp. (Jafari and Behroozian 2010); genus Iris (Xiao Fang et al. 2009) and Daucus spp. (Lovene et al. 2008, Xiao Fang et al. 2009). Although karyotype does not look at genes but chromosomes, it provides useful information for an effective plant improvement program. Such information can be used in the characterization of plant agronomic performance, determination of origin and evolution pathway of new species, re- vealing the possible occurrence of genetic disorders and chromosomal abnormalities (Claire 2008).

Materials and methods

Plant materials G. latifolium accessions used in this study were collected from humid forest vegetation of Abia, Akwa Ibom, Cross River and Imo states of Nigeria (Table 1). Vines of accessions were obtained from adult plants and preserved till they were replanted at the Spice Field Genebank located behind the Biological Sciences building University of Calabar (Long. 008°21′E, Lat. 04°47′N 4°56′N, 155 m above sea level). These were planted on on ridges at a spacing of 30 cm between plants and 50 cm between ridges and vines were supported with upright stakes. Weeding and other cultural operations were carried out manually.

Karyotype analysis Root tips of Gongronema latifolium were collected between 7:00–11:30 a.m. Immediately

Table 1. Collection sites of G. latifolium accessions from humid forest vegetation of Nigeria

Village LGA State Latitude °N Longitude °E Altitude (m)

Umudike-Uku Ikwuano Abia 04.8604 7.7843 121 Ikot Udobia Etinan Akwa Ibom 04.8863 7.8299 67 Iko-Ekperem Akamkpa Cross River 05.6068 08.2170 10.32 Umuogba Ntu Ngor Okpala Imo 04.9975 08.3330 45.3 2012 Karyotype and Ploidy Level in Gongronema latifolium 45 after harvesting, root tips were pretreated with 8-hydroxyquinoline and kept at room temperature for 3 h. The pretreated root tips and buds were rinsed in distilled water and fixed in a cold mixture of ethanol and acetic acid (3 : 1). Fixed samples were used after 12–24 h or transferred to 70% alco- hol and stored in refrigerator until required. The fixed materi als were hydrolyzed in 1N HCl at 60°C for 5–8 min. Meristematic portions were cut off onto clean slides in a drop of formic lacto propionic (FLP) orcein stain. The meristem was gently tapped with a squashing rod. A cover slip was placed over the top and the excess stain was removed with filter paper by applying a firm thumb pressure. The slides were sealed with nail hardener. Prepared slides were viewed under the microscope at a magnification of ×100. Photographs were taken of chromosomes at the metaphase stage using a Canon Power Shot A630, 8.0 megapixel digital camera with 4× Optical Zoom. The chromosomes were described based on size, arm length, relative length, haploid set length, ratio, number and shape of chromosomes (Vargas et al. 2007, Levan 1964). Measurements of chromosomes were carried out using an oculometer (R1370-19) calibrated with a stage micrometer at 0.77 μm for each unit of the oculometer.

Ploidy determination by flow cytometry( FCM) Nuclear samples were prepared from intact young vine stem cutting of Gongronema latifolium chopped in a glass Petri dish with new razor blade in 1.0 ml of ice cold nucleic isolation buffer from DNA kit (Cysteine UV, P. Partec GMBH, Germany). The crude suspension was incubated at room temperature for 5 min and then filtered through a 30 μm filter and stained with 4,6-diamidino-2-phe- nyl indole (DAPI). Samples were incubated for 10–15 min period after which they were analyzed in a Partec PA 11 flow cytometer equipped with an air-cooled argon-ion laser (JDS Uniphase, San José, CA, USA) operating at 488 nm. Fluorescence histogram depicting intensity of isolated nuclei was plottedon a semi-logarithmic scale. At least 5,990 nuclei were analysed per sample. For each sample, the percentage of nuclei present in each peak was calculated using the SYSTEM II v.3.0 software (Beckman Coulter, Hialeah, FL, U.S.A.). Ploidy level was determined by comparing the position of dominant peaks corresponding to nuclei at G0–G1 phase of the cell cycle.

Results

Karyotype analysis The number of somatic chromosomes along with the details of the karyotypes of the studied accessions of Gongronema latifolium is shown in Tables 2 and 3. The results indicated variation in the chromosome number of G. latifolium from the different accessions studied.

Cross River accessions (2n=18, 2n=20) The karyotype of the Cross River accession, 2n=18 (Plate 1a, Fig. 1a), is shown in Table 2. The ideogram of this karyotype showed 2 pairs of chromosomes were metacentric (chromosomes 5 and 8) with arm ratio 1.0. Four pairs of chromosomes were sub-metacentric (chromosomes 2, 4, 6 and 7) having arm ratios of 1.5, 1.7, 1.3 and 2.0, respectively. Two pairs were acrocentric, chromosome numbers 1 and 3, with arm ratios of 4.0 and 3.0, respectively. One pair of chromosomes, that is, chromosome number 9, was telocentric. The mean length of these chromosomes (2.87 μm) ranged from 1.54 μm in chromosome 9 to 3.85 μm in chromosomes 1 and 2. The haploid set length (HSL) was 25.82 μm (Table 2). The largest chromosome (chromosomes 1 and 2) represented 14.9% each of the total genome while the shortest chromosome (chromosome 9) represented 6.0%. In the Cross River accession with 2n=22 (Plate 1b, Fig. 1b), the karyotype showed that 3 pairs of chromosomes were metacentric (chromosomes 5, 6 and 9) with an arm ratio of 1.0. Four pairs of chromosomes were sub-metacentric (chromosomes 1 and 2) with an arm ratio of 1.3 each and chromosomes 7 and 10 with arm ratios of 1.5 and 2.0, respectively. Three pairs of chromosomes were 46 P. O. Aikpokpodion et al. Cytologia 77(1)

Plate 1. Mitotic metaphase chromosomes of the Cross River accession with (a) 2n=18 (mag. ×8000) and (b) 2n=20 (mag. ×6000)

Fig. 1. Ideogram of chromosomes obtained from the Cross River accession (a) 2n=18 (b) 2n=22

Plate 2. Mitotic metaphase chromosomes of the Abia accession with (a) 2n=20 (mag. ×5000) and (b) 2n=22 (mag. ×5000) acrocentric (chromosomes 3, 4 and 8) with arm ratios of 2.0, 3.5 and 5.0, respectively. One pair of chromosomes (chromosome 11) was telocentric. The mean length (3.75 μm) of these chromosomes ranged from 2.31 μm in chromosomes 10 and 11 to 5.39 μm in chromosomes 1 and 2. The haploid set length was 41.29 μm (Table 2). The relative length of the largest chromosome (chromosomes 1 and 2) represented 13.05% and the shortest chromosome (chromosomes 10 and 11) represented 5.59% of the entire genome.

Abia accessions (2n=20, 2n=22) The karyotype formula of the Abia accession, 2n=20 (Plate 2a, Fig. 2a), is shown in Table 2. In this karyotype, 3 pairs of chromosomes were metacentric (chromosomes 6, 7 and 9) with an arm ratio of 1.0. Four pairs of chromosomes were sub-metacentric: chromosome 1 with an arm ratio of 1.3; chromosome 3 and 4 with an arm ratio of 1.5 and chromosome 8 with an arm ratio of 2.0. Two 2012 Karyotype and Ploidy Level in Gongronema latifolium 47

Fig. 2. Ideogram of chromosomes obtained from the Abia accession (a) 2n=20 (b) 2n=22

Table 2. Karyotype formulae and karyomorphological description of chromosomes of G. latifolium

Accession 2n L S T RL R HSL Formula

Abia 20 2.2 1.2 3.4 10.0 1.6 33.9 3m+4sm+2a+1t 22 2.2 1.1 3.3 9.1 1.4 35.8 2m+4sm+2a+3t Akwa Ibom 18 2.1 1.4 3.5 11.1 1.8 31.6 3m+5sm+1a Cross River 18 1.9 1.0 2.9 11.1 1.7 25.8 2m+4sm+2a+1t 22 2.4 1.4 3.8 9.1 1.8 41.3 3m+4sm+3a+1t Imo 22 3.5 1.9 5.4 9.1 1.3 59.3 1m+5sm+3a+2t 24 3.6 2.7 6.3 8.3 1.3 75.1 2m+7sm+2a+1t

L: Long arm, S: short arm, T: total length, HSL: haploid set length, RL: relative length, R: ratio pairs of chromosomes were acrocentric (chromosomes 2 and 5) with arm ratios of 4.0 and 3.0, respectively. One pair of chromosomes, chromosome 10 was telocentric. The mean length (3.39 μm) of the chromosomes ranged from 2.31 μm in chromosome 8 to 5.39 μm in chromosome 1. The haploid set length was 33.89 μm (Table 2). The relative length of the largest chromo some (chromosome 1) represented 15.90% of the total genome and the shortest chromosome (chromosome 8) represented 6.82%. In the Abia accession with 2n=22 (Plate 2b, Fig. 2b), the karyotype showed that 2 pairs of chromosomes (chromosomes 4 and 7) were metacentric with an arm ratio 1.0. Four pairs of chromosomes were sub-metacentric: chromosome 1 with an arm ratio of 1.3, chromosomes 2 and 8 with arm ratios of 2.0 and chromosome 5 with an arm ratio of 1.5. Two pairs of chromosomes were acrocentric (chromosomes 3 and 6) with arm ratios of 4.0 and 3.0, respectively. Three pairs of chromosomes were telocentric (chromosome 9, 10 and 11). The mean length (3.26 μm) of the chromosomes ranged from 1.16 μm in chromosome 11 to 5.39 μm in chromosome 1. The haploid set length was 35.81 μm (Table 2). The largest chromosome (chromosome 1) represented 15.05% of the total genome while the shortest chromosome (chromosome 11) represented 3.24% of the total genome.

Akwa Ibom accession (2n=18) Eighteen pairs of chromosomes were observed at the metaphase stage (Plate 3), 2n=18. The ideogram (Fig. 3) constructed for the karyotype (Table 2) of the Akwa Ibom accession showed that 3 pairs of chromosomes were metacentric (chromosomes 3, 6 and 7) with an arm ratio of 1.0. Five pairs of chromosomes were sub-metacentric: chromosomes 1, 8 and 9 with arm ratios of 2.0 each, and chromosomes Plate 3. Mitotic metaphase chromosomes 4 and 5 with arm ratios of 1.5 each. One pair of of the Akwa Ibom accession chromosomes, chromosome 2 with an arm ratio of with 2n=18 (mag. ×8000) 48 P. O. Aikpokpodion et al. Cytologia 77(1)

4.0, was acrocentric. The mean length (3.51 μm) of the chromosomes ranged from 2.31 μm in chromosomes 8 and 9 to 4.62 μm in chromosome 1 and 3. The haploid set length was 31.57 μm (Table 2). The relative length of the largest chromosome (chromosomes 1 and 3) represented 14.63% of the total genome while the relative length of the shortest chromosome (chromosomes 8 and 9) represented 7.32%.

Imo accession (2n=22, 2n=24) Variation in chromosome number was observed among plants in this accession. Chromosome numbers of the metaphase stage were 2n=22 and 24 (Plates 4a and 4b). The ideogram constructed based on the chromosome size (short arm length and long arm length) for the 2 numbers observed are shown in Figs. 4a and 4b. Fig. 3. Ideogram of chromosomes ob- For the Imo accession, 2n=22, the karyotype tained from the Akwa Ibom accession 2x=18 (Table 2) showed that 1 pair of chromosomes (chromosome 8) was metacentric with an arm ratio of

Table 3. Karyotype form in percentages in 4 G. latifolium accessions used in this study

Chromosome form Cross River Abia Akwa Ibom Imo 2n 18 22 20 22 18 22 24

Metacentric (%) 22.2 27.3 30.0 18.2 33.3 9.1 16.7 Submetacentric (%) 44.4 36.4 40.0 36.4 55.6 45.5 58.3 Acrocentric (%) 22.2 27.3 20.0 18.2 11.1 40.9 16.7 Telocentric (%) 11.1 9.1 10.0 27.3 ̶ 18.2 8.3

Plate 4. Mitotic metaphase chromosomes of the Imo accession with (a) 2n=22 (mag. ×5000) (b) 2n=24 (mag. ×6000)

Fig. 4. Ideogram of chromosomes obtained from the Imo accession (a) 2n=22 (b) 2n=24 2012 Karyotype and Ploidy Level in Gongronema latifolium 49

1.0; 5 pairs were sub-metacentric: chromosomes 2, 3, 5 and 9 with an arm ratio 1.5 each, and chromosome 6 with an arm ratio of 1.4. Three pairs of chromosomes, chromosomes 1, 4 and 7, were acrocentric with arm ratios of 1.8, 2.5 and 2.3, respectively. Two pairs of chromosomes, chromosome 10 and 11, were telocentric. The mean length (5.39 μm) of the chromoso mes ranged from 3.08 μm in chromosome 11 to 9.63 μm in chromosome 1. The haploid set length was 59.32 μm (Table 2) and the largest chromosome (chromosome 1) represented 16.23% of the total genome while the shortest chromosome (chromosome 11) represented 5.19%. The karyotype (Table 2) of Imo accession, 2n=24 showed that 2 pairs of chromosomes were metacentric (chromosomes 4 and 7) with an arm ratio of 1.0. Seven pairs of chromosomes were sub-metacentric: chromosomes 1, 2 and 3 with an arm ratio of 1.2 each, chromosomes 5, 8 and 9 with an arm ratio of 1.3 each and chromosome 11 with an arm ratio of 1.5. Two pairs of chromosomes (chromosomes 6 and 10) were acrocentric with an arm ratio of 1.7 and 2.7, respectively. One pair of chromosomes, chromosome 12, was telocentric. The mean length (6.26 μm) of the chromosomes ranged from 2.31 μm in chromosome 12 to 10.0 μm in chromosome 1. The haploid set length

Fig. 5. Flow cytometry evalustion of ploidy level in 4 accessions of Gongronema latifolium. Histograms of relative fluorescence intensity of nuclei isolated from stem cuttings of the (a) Abia accession (b) Akwa Ibom accession (c) Cross River accession (d) Imo accession. The haploid genome size (C) is defined as the nuclear DNA content of the gamete. The nuclei are defined as having 2C DNA content for somatic cells in GI phase and 4C DNA content for those in G2 phase. 50 P. O. Aikpokpodion et al. Cytologia 77(1)

Table 4. Flow cytometry data for 4 G. latifolium accessions used in this study

Accessions Cross River Abia Akwa Ibom Imo

Peaks 1C 2C 4C 1C 2C 1C 2C 1C 2C

Index 1.0 2.1 4.2 1.0 2.1 1.0 2.1 1.0 2.0 Mean 48.5 99.6 202.3 45.5 98.3 46.8 96.5 48.5 98.9 Area 2320 1044 139 2444 1182 1699 1632 2173 817 Area (%) 38.3 17.2 2.3 40.7 19.7 28.1 27.0 36.2 13.6 CV (%) 2.6 2.8 1.9 3.9 2.9 3.7 2.3 2.6 4.3

Total count 6060 6013 6046 5996

Gain (lin) 381.0 388.0 381.0 381.0 was 78.05 μm and the largest chromosome (chromosome 1) represented 13.33% of the total genome while the shortest chromosome (chromosome 12) represented 3.08%.

Flow cytometric analysis Pherograms obtained from ﬂow cytometry analysis (Fig. 5) indicated 2 dominant peaks at channels 50 and 100 for all accessions from Abia, Akwa Ibom, Cross River and Imo. However, a third peak was obtained at channel 200in Cross River accession. The percentage of cells with haploid genome size (1C), that is nuclei DNA content of the gamete, ranged from 28.1% in the Akwa Ibom accession to 40.7% in the Abia accession (Table 4). Somatic cells in G1 phase (2C DNA content) ranged from 13.6% for the Imo accession to 27.0% for the Akwa Ibom accession. Only in the Cross River accession were cells at the G2 phase with nuclei having 4C DNA content observed in 2.3% of counts.

Discussion

In this study, ploidy level and karyotype analyses were carried out to provide basic genetic information on the African bitter milk plant (Utazi), Gongronema latifolium. Karyotype analysis indicated the occurrence of 4 basic numbers (n=9, 10, 11 and 12). While one chromosome number, 2n=18, was obtained for the Akwa Ibom accession, 2 chromosome numbers each were obtained for the Cross River accession (2n=18 and 22), the Abia accession (2n=20 and 22) and the Imo accession (2n=22 and 24). Since there is no previous report on the cytology of the genus Gongronema, the reported basic chromosome number in the family Asclepiadaceae presented the closest basis for comparison. The reported basic chromosome number for the family Asclepiadaceae was n=(8–), 11 (or 12) (Tsiang and Li 1977, GBIF 2007). While we obtained a similar chromosome number as reported for some members of the plant family Asclepiadaceae (GBIF 2007) in some cytotypes, a new number, n=9, was found in the Akwa Ibom and Cross River accessions while n=10 was found in the Abia accession. We therefore conclude the presence of cytotype variation in G. latifolium, and, that at least 4 cytotypes (n=9, 10, 11 and 12) were present in the humid forest vegetation where these species are endemic. The occurrence of cytotypes was also reported in Iris species (Xiao Fang et al. 2009) and Borago species (Selvi et al. 2006). This variability in chromosome number of the accessions may also be due to geographical background (Allopatry). Variation was detected between and within the accessions for content of DNA assessed by measuring the haploid set length and chromosome relative length. The Cross River accession has the smallest chromosomes while the Imo accession has the largest chromosome size. This showed phylogenetic advancement in the Cross River accession in terms of asymmetry of chromosomes accompanied by reduction of chromosome number. Results from haploid set length and chromosome 2012 Karyotype and Ploidy Level in Gongronema latifolium 51 relative length indicated that an increase in chromosome length is accompanied by an increase in relative length leading to an increase in proportion of the entire genome represented by the chromosome (Vargas et al. 2007). The karyotype formulae indicated rearrangements within chromosomes and showed a tendency for asymmetry. The known predominant trend in plant karyotype evolution is from symmetry to greater asymmetry Stebbins (1971). Symmetrical karyotypes are usually found in more primitive types of a species. With evolution being a continuous process, it can be con- cluded that evolutionary process is still very active in Gongronema latifolium, an indigenous species to west African tropical forest. Flow cytometry study indicated that cells were in both gametic (S) and somatic (G1) phases of the cell cycle as indicated by the 1C and 2C DNA content, respectively. This confirmed G. latifolium is a diploid plant species. However, the observation of a third peak indicating 4C DNA content in the Cross River accession provided evidence for either cells at the G2 phase, a second period of cel- lular growth prior to the next point of mitosis or endoreduplication. Endoreduplication involves re- peated cycles of DNA synthesis without occurrence of cell divisions, and it is a common phenome- non in differentiated cells of seed plants (Castro et al. 2007, Smulders et al. 1995). This process leads polysomaty, defined as the presence of cells with various ploidy levels in an organ (Sliwinska and Lukaszewska 2005). The occurrence of endoreduplication in Gongronema latifolium is suggested from the flow cytometry evidence. However, more studies will be needed to establish this. Given that the DNA sample was obtained from stem cuttings which include organs such as phloem and xylem for the transport of nutrients and water as well as plant stability, the occurrence of polysomaty could not be dismissed. Although the mechanism of endoreduplication has not been thor- oughly investigated, it is found extensively in tissues reserved for nutrients uptake and storage such as plant leaves and root hairs (Kondorosi et al. 2000) and tissues that are needed to maintain ho- meostasis (Menand et al. 2007). Evidence of endoreduplication in G. latifolium as suggested in this study could therefore be regarded as an intrinsic survival mechanism in this climber species that thrives both in dense under storey forest and unshaded conditions in its native habitat.

Acknowledgements

The authors thankfully acknowledge the World Bank assisted STEP B project grant award for ‘Conservation of Spice plants in Nigeria’ within which this study was conducted. We also acknowledge the support of Ikootobong Urua, Dr. (Mrs.) Osuagwu, Professor Ene-Obong and Dr. Valentine Ntui, all of the Department of Genetics and Biotechnology, University of Calabar.

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