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Annals of Biological Research, 2011, 2 (4) : 85-90 (http://scholarsresearchlibrary.com/archive.html)

ISSN 0976-1233

CODEN (USA): ABRNBW

85 Scholars Research Library

Mitotic Chromosomes in Abelmoschus esculentus (L.) Moench esculentus (L.) Moench

C. C. Nwangburuka1*, O. B., Kehinde2, O. A. Adegbite2, O. A. Denton1

1Department of Agriculture, Babcock University, Ilishan-Remo, Ikeja, Lagos, Nigeria

2Department of Plant Breeding and Seed Tech. University, of Agricultue Abeokuta, Abeokuta, Nigeria ______________________________________________________________________________

ABSTRACT The mitotic chromosomes of A. esculentus was investigated using twenty nine accessions sourced from the genetic resource units of five teaching and research institutions in Nigeria. Mitotic chromosomes were prepared using Eiosin stain on a glass slide and viewed on Nautica microscope at X400 and X1000. Using the motic image 2000.2.0 computer programme, chromosome size was determined. All the accessions had identical chromosome number of 2n = 130. There was uniformity in the morphology of the chromosomes. A few were assymmetrical or sub-metacentric, while majority were symmetrical or metacentric . Chromosome length ranged from 3.0 µm to 16 µm and classified into eight groups. Mitotic division was at the peak in most of the accessions at about 16:00hr, 24:00hr and 3:00hr. This suggests that mitotic counts at these periods will give better result. Key words: mitotic dynamics, chromosomes, okra, sub-metacentric. Abbreviations: BU: Babcock University, GRU: Genetic resource unit ______________________________________________________________________________

INTRODUCTION The earliest reports on chromosome size and shape in A. esculentus was by Datta and Naug [1]. These authors classified A esculentus chromosomes into eight types (A, B, C, D, E, F, G, H) based on chromosome length. Chromosome length decrease from “Type A” to “Type H”Chromosome length ranged from 3 µ to 0.75 µ. There is a wide variation in chromosome number in the genus Abelmoschus, and which may be attributed to the difficulty experienced in counting because of high chromosome numbers. Large chromosome numbers in okra has been suggested to provide excellent opportunity for very wide recombination [2]. The frequently reported chromosome numbers for A. esculentus range between 2n = 66 and 144. Other reports include 2n = 124 [3], 2n=130 [4], 2n = 131 - 143 [5] and 2n = 144 [1], 2n= 130-140 [6]. The evidence from several reports confirm that A. esculentus exists more stably as a diploid species with 36 pairs of chromosomes (2n = 2x = 72) [7] [8]. However, A. esculentus is reported to

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exists as a tetraploid with chromosome number 2n = 4x = 144 [1] [9], thus implicating polyploidy in the evolution of Abelmoschus species. However, the frequently observed chromosome number, however, is 2n= 130, although, Datta and Naug (1968 (1)) suggested that the numbers 2n = 72, 108, 120, 132 and 144 reveals gradual increase in ploidy with x 12. Consequently Hamon, [10] suggested two kinds of Abelmoschus genotypes 2n = 60 – 70 as diplods a diploid and 2n = 120 - 130 as tetraploids. However, Joshi and Hardas [11] and IBPGR [12] claimed A. esculentus as an amphidiploids of A. tuberculatus (2n = 58) and A. ficulneus (2n = 72). Datta and Naug [1] opined that these variations in chromosome number could be due to irregularities in chromosome movement during mitotic phase of cell division. However, Akhond and Molla [13] reported that A. esculentus germplasm exhibited possible inter-specific cross compatibility with their wild relatives, but they are usually characterized by pre- and post-zygotic barriers. However, the extent of out-crossing was observed to vary between 0 and 60 % depending on variety, the cropping season and/or the location [13]. This seems to confirm the possible source of the different chromosome numbers of A. esculentus. Meanwhile, Chromosome studies require an understanding of the mitotic dynamics(mitotic trend) of the cell. This provides the best time to fix cells (root-tip cells ) for accurate chromosome count and morphology could be visible and easily determined. Early reports confirm metaphase as the appropriate time to study chromosome morphology and number [14] [15] [16]. There is therefore the need to ascertain the exact chromosome number and morphology for A. esculentus accessions distributed in this okra growing region and also determine the best time to fix roots for effective cytological studies. This information will be vital in okra characterization and breeding to ensure for proper cross compatibility and stability in chromosome number. Hence, this study will determine the exact chromosome number of twenty nine cultivated A. esculentus accessions and consequently elucidate species mitotic trends

MATERIALS AND METHODS

Twenty nine (29) accessions of A. esculentus were sourced from germplasm facilities maintained at the Genetic Resource Units (GRU) of five academic and research institutions. Sixteen accessions were from the National Horticultural Research Institute (NIHORT) Ibadan, five (5) from University of Agriculture, Abeokuta (UNAAB), four (4) from Babcock University Ilishan-Remo (BU), four (4) from National Centre for Genetic Resources and Biotechnology (NACGRAB) Ibadan and one line (1) from the Institute of Agricultural Research and Training (IAR&T) Ibadan (Table 1). Seed germination. One hundred viable seeds were selected from each of the twenty-nine accessions and grown in Petri-dishes lined with filter paper and cotton wool and soaked with distilled water. After root emergence (2 – 3 days after), they were harvested at 1 h interval in 24 h cycle using sharp razor and forceps. Pre-fixation and Fixation of root Roots were pre-fixed in 0.04% colchicine for 24 hours to dissolve the spindle fibres and immobilise the chromosomes at the different stages. The Pre-fixed roots were rinsed in distilled water, fixed in 1:3 acetic ethanol for 24 hours and stored at 4 OC until ready for use.

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Microscopic Observation The root were removed from 1:3 acetic ethanol after 24 h, rinsed in distilled water and hydrolyzed in 1N HCl (18% HCl) for 4 minutes. Hydrolyzed root-tips were squashed on a glass slide and stained with a few drops of FLP-Orcein. Chromosome count Prepared slides were viewed using a phase contrast microscope. Cell observation for mitosis was done at X400. Chromosome count and photomicrographs were taken under oil immersion at x1000 using the motic image 2000 2.0 version computer programme.

RESULTS

all the accessions had 2n = 130. There was uniformity in the morphology of chromosomes. A few were sub-metacentric, while majority were symmetrical. Chromosome length ranged from 3.0 µm to 16 µm and were classified into eight groups. There was evidence of slight cytoplasmic staining in some cells of few accessions, but did not interfere significantly with microscopic observations and chromosome count There was considerable evidence that mitotic divisions among the accessions were continuous but varied in intensity. The result of the analysis of variance at P = 0.01% showed significant difference in all the mitotic phases with respect to time. Mitotic activities in cells were considerably low between 6:00hr and 12:00hr as evident in the increased percentage of interphase. This rose significantly at 16:00hr as indicated by the increase in prophase and metaphase. The number of cells at metaphase gradually decreased while those at prophase increased until 19:00hr when it exceeded all other mitotic phases. The phases of mitosis took different trends except for anaphase and telophase, whose trends were similar. Thus, there was an apparent synchrony in their cycle as depicted in the peaks of their curves. Mitotic activities in cells gradually decreased but increased number of cells was observed at interphase. This lowering of mitotic activities was consistent until 24:00hr, after which activities resumed in most cells, and by 03:00 h more cells had entered into metaphase. There was a significant decrease of mitotic activities, and with more number of cells remained at interphase. Thus, mitotic assessment is advised between 24:00hr and 3:00hr. The number of cells at prophase remained high to moderate and decreased from 9:00hr and 8:00hr. By 17:00hr, the number of cells at prophase exceeded those at interphase and the other phases, and from 20:00hr a new mitotic cycle had began and cells were all at interphase.

DISCUSSION

The analysis of variance indicates significant difference in the rate of mitotic phases in respect to time periods. This is an indication that the root cells of A. esculentus have different periods when growth was optimised. The prevalence of Interphase seems to confirm the reports of Francesca [15]. The intermittent increase and decrease in the frequency of mitosis observed in short time intervals suggests that cell division is dynamic in okra and may be short lived. There is also a probability of more than one cycle of mitosis within 24 hours in this species. The low rate of mitosis observed between 6:00 am and 12:00 noon agrees with Davie [14]; suggesting a low physiological activity in cells. The number of prophase and interphase remained relatively high throughout the 24 hour period. This may probably be because prophase generally takes most of the time in the cell cycle [16]. Furthermore, interphase has been reported by Francesca [15] as a period of DNA synthesis and replication which probably requires a longer period of time in the

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cell cycle. Most of the accessions had their peak metaphase periods at 16:00hr, 24:00hr and 3:00hr. This implies that mitotic activities in okra cell are optimum at these periods and suggests that root tips fixed at these times for chromosome studies will give better result. This result agrees with earlier work by Davie [14], who reported that plates suitable for chromosome count can be obtained when roots are fixed between 23:00hr and 2:00hr in the family Malvaceae. However, the highest metaphase rate was observed at 24:00hr and 3:00hr in most of the accessions. The chromosome number of the accessions studied was 2n = 130. This agrees with earlier reports of [1] [6] and [5]. Similarly, the chromosome size of between 3 µm to 16 µm observed in this study was at variance with the report of Datta and Naug [1] although, chromosomes were in eight groups based on chromosome length. The considerable high uniformity in the morphology of the chromosomes expressed in a few that were sub-metacentric and the majority that were symmetrical agrees with the report of Datta and Naug, [1]. When chromosomes were stained with FLP orcein as reported in Musa spp. [17], there was slight cytoplasmic staining observed in cells due to chromatin associated structures in the cytoplasm of root tip cell, since FLP orcein is chromatin- specific [18].

Table 1: Accessions and their sources

Serial Number Accession Name Source

1 Lady’s Finger UNAAB

2 OLA KA-1-6-05 NIHORT

3 OLA V1 NIHORT 4 OLA K2005 NIHORT 5 NIHORT Ilagidi UNAAB

6 LD88/1-8-11-1 NIHORT 7 LD88/1-8-5-2 NIHORT 8 Short Mouth Ibarapa UNAAB 9 Clemson spineless NACGRAB 10 V45-2 NIHORT

11 NH99/DA NIHORT 12 LD88/1-8-16-2 NIHORT 13 OLA 99/13 NIHORT

14 OSADEP Purple Tall UNAAB 15 47-4-5 NIHORT 16 ENUGU-1 NACGRAB 17 47-4 NIHORT 18 V2-OYO UNAAB

19 V-35 IAR&T

20 OLA 3 LOCAL NIHORT

21 OK 20 NIHORT 22 NH99/28 NIHORT 23 Dajofolowo 1 BU 24 CCN2005/2 BU 25 NH88/1-8-16-2 NIHORT

26 NH88/82 NIHORT 27 NH99/9 NIHORT 28 Jokoso 2 BU 29 CCN2005/1 BU

NIHORT: National Horticultural Research Institute,Ibadan, UNAAB: University of Agriculture, Abeokuta, BU: Babcock

University Ilishan-Remo, NACGRAB: National Centre for Genetic Resources and Biotechnology, IAR&T: Institute of Agricultural Research and Training, Ibadan.

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A. 2n=130, LD88/1-8-11-1 B. 2n= 130, V2 OYO C. 2n=130, Enugu-1 D. 2n=130, Dajofolowo E. 2n= 130, CCN2005/2 F. 2n=130, Jokoso G. 2n= 130, Ila gidi H. 2n= 130. Lady’s finger

Figure 1: Meiotic and Mitotic chromosomes in okra accessions at metaphase

Figure 2: Rate of mitosis in okra within the period of 24 hours

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