Polyploidy Expressions of Colchicine Induced Maize Genotypes
Olawuyi Odunayo Joseph1* and Adeosun Chidiebere Anastacia2
1Department of Botany, Genetics and Molecular Biology Unit, University of Ibadan, Nigeria
2Department of Plant Science and Biotechnology, University of Nigeria, Nsukka, Nigeria
*Correspondence to: Olawuyi Odunayo Joseph
Citation: Joseph OO, Anastacia AC (2018) Polyploidy Expressions of Colchicine Induced Maize Genotypes. SCIOL Biotechnol 2018;1:72- 82
The polyploidy expressions of colchicine mutagen at concentrations of 0.05%, 0.1%, 0.15%, 0.2%, 0.3%, 0.5% and 0.7% were investigated on maize genotypes. The in-vitro and field experiments were carried out at the Genetics and Molecular Biology laboratory and Research Farm of the Department of Botany, University of Ibadan, Nigeria. The concentration and genotype had significant (p ˂ 0.05) effect on percentage emergence, though increased concentration showed a lethal effect on DTSR-WC and SAMMAZ19S-14 DRD genotypes. The mutagen showed significant lethal effect on the percentage emergence of plants as the concentration of colchicine increased. SAMMAZ19S-14 DRD at 0.15% and TZECOMP3C2 at 0.1% showed higher ploidy with high coefficient of variation of 4.36 and 4.38. However, ACR.91.SUWANI-SRC exhibited mixoploid at 0.1% concentration of colchicine though, remained unaffected by other concentrations, but found to be more tolerant genotype to mutagenic effect.
Colchicine, Maize genotypes, Morphology, Ploidy
Maize (Zea mays L.) is an annual cereal grown world-wide. It serves as a source of nutrients for animals and basic raw material for industrial products such as; starch, oil and protein, alcoholic beverages, food sweeteners and, more recently, fuel [1-3]. The monoecious nature of maize has helped in its development by human and as a result of its large amount of hybrid vigor, its genetic improvement had been successfully achieved through breeding . Plant breeding deals with the identification and utilization of genetic variation.
Mutation breeding is an effective tool used by plant breeders for genetic improvement of autogamous crops with narrow genetic base and developmental phenomena . It alters one or two major desired agronomic traits which limit their productivity or enhance their quality [6,7]. Polyploidy is an important genomic feature found in eukaryotes, particularly in plants. It played key role in plant speciation, diversification, evolution and breeding which leads to variation in plant phenotype [8,9].
Polyploid plants can be obtained through the use of colchicine, which is an alkaloid and a chemical mutagen contained in seeds and bulbs of Colchicum autumnale L. [10,11]. According to [9,11,12], polyploidy plants differ from their progenitors in morphological, ecological, physiological, and cytological characteristics which include; broader leaves, good quality, high-yielding and enhanced resistance to environmental stress and diseases. In spite of limited information on polyploidy expressions in plants, this study therefore, investigated the polyploidy expressions of maize induced by colchicine.
Materials and Methods
Germplasm collection of seed samples, experimental location and research design
The seeds of maize collected from germplasm unit of the International Institute of Tropical Agriculture (IITA), Ibadan and the Institute of Agricultural Research and Training, Nigeria (IAR&T) were; SAMMAZ19S-14DRD, TZE COMP3C2, ACR.91.SUWANI-SRC, DTSR-WCO.28 and DSTR-WC.
The in vitro experiment was carried out at the Genetics and Molecular Biology laboratory of the Department of Botany, University of Ibadan, Nigeria. The screen house and field experimental were also carried out at the research farm (Longitude 7.417ºN and Latitude 3.900ºE) of the Department of Botany, University of Ibadan. The experiment was laid out in complete randomized design with three replicates.
Colchicine preparation, seed pre-treatment and in vitro screening of maize genotype induced with colchicine
The different concentrations of colchicine used (0.05%, 0.1%, 0.15%, 0.2%, 0.3%, 0.5% and 0.7%) were prepared according to the method described by . The seeds were pre-soaked in different concentrations for 16 hours according to the method described by , while pre-soaked seeds in distilled water served as control.
The seeds were then rinsed with sterile water and planted on Petri dishes lined with moist filter paper. Three seeds of each maize genotype were planted into each Petri dish. Seeds were observed for growth and survival rate.
Screen house and field experiments
The pre-soaked seeds of selected genotypes in concentrations of colchicine (0.05%, 0.1%, 0.15% and 0.2%) were evaluated in screen house. The seeds treated in concentrations (0.1%, 0.3%, 0.5% and 0.7%) were evaluated in field experiments while seeds treated distilled water served as control. After 16 hours, seeds were rinsed with sterile water before planting. The polythene bags were spaced at distance 30 cm, while the three replicates per genotype were separated at the distance of 40 cm. The survival and growth characters of seeds such as: plant height, number of leaves, stem girth, leaf length and leaf width were determined.
Determination of ploidy level
The ploidy level was determined using flow cytometry according to the method described by Ochatt .
Sample preparation was carried out using a sterile Petri dish and 500 µl of OTTO 1 solution (0.1 M citric acid + 0.5% (v/v) tween 20) was pipetted carefully into it. A piece of a freshly cut young plant tissue (leaf) was placed in the Petri dish and chopped to small pieces using sharp razor blade.
Another 500 µl of the OTTO 1 solution was added slowly to wash the leaf chopped downward in a slanted position. The mixture was passed through a filter (0.22 nM) into a clean tube and allowed to incubate for two minutes at room temperature.
1 ml of the OTTO 11 (400 mM Na2PO4.12H2O (pH 8-9)) working solution was added and thoroughly mixed; the ploidy level was determined using a flow cytometer.
Data were analysed for Analysis of variance (ANOVA) using Statistical Analysis Software (SAS version 9.1). Differences in means were separated by Duncan Multiple Range Test (DMRT) at 95% probability level (p ˂ 0.05).
Results and Discussion
The effect of colchicine treatments on percentage emergence of maize genotypes is shown in Table 1. The percentage emergence of in vitro treated plant at 0.3% and untreated (control) plant on the field are significantly (p < 0.05) higher than other treatment levels. There is no significant difference between the control and 0.1% colchicine treatment for both emerged in-vitro and field plants. The least percentage of emergence was recorded for both in vitro and field plants treated at 0.5% (20.00%, 4.44%) and 0.7% (15.56%, 6.67%) respectively. This revealed that higher doses of the mutagen exert more inhibitory effect when compared to the lower doses which confirmed the observation made by .
The significant difference in genotypic performance of emerged plants and growth characters indicated variation among the genotypes, which supported the findings made by .
ACR.91.SUWANI-SRC genotype is significantly higher and different in mean percentage emergence (46.67%), plant height (6.12 cm), number of leaves (3.40), stem girth (1.37 cm), leaf width compared to DTSR-WC and SAMMAZ19 S-14 DRD which had the least performance of percentage emergence and growth characters of maize (Table 2). This could be due to its high mutagenic tolerance to the treatments in agreement with the findings of [16,18]. Higher mortality rates of plants with respect to higher colchicine concentrations had also previously reported by .
Table 2: Mean effect of genotypes on percentage emergence and growth characters of maize. View Table 2
The ploidy result of the colchicine induced maize genotypes shown in Table 3 revealed that the genotypes were originally tetraploid as revealed by the control which could be as a result of its natural tetraploid origin which confirmed the findings of [20,21]. TZE COMP3C2 treated with 0.1% concentration of colchicine had the highest coefficient of variation (CV) of 4.38, while 0.1% treatment of SAMMAZ19 S-14 DRD had the highest mean. The CV of SAMMAZ19 S-14 DRD treated with 0.15% colchicine was higher than other treatment of the same genotype. 0.1% of colchicine treated with ACR.91.SUWANI-SRC showed two different means and two different CV, One mean was found to be twice as high as the second indicating the occurrence of the duplication of the chromosome. The variation in coefficient of variation of the same genotype is due to the effect of the different concentrations of the mutagen. The result revealed that the effects of colchicine on the genotypes are not strong enough to induce polyploidy except for genotype ACR.91.SUWANI-SRC at 0.1% colchicine concentration, which exhibited mixoploid supporting similar polyploidy effect of colchicine confirmed by [10-12,22].
Figure 1, Figure 2, Figure 3, Figure 4, Figure 5, Figure 6, Figure 7, Figure 8, Figure 9 and Figure 10 showed the ploidy histogram of the induced maize genotypes. The result showed that maize genotypes had duplicated chromosomal segments naturally apart from the treatment effect. This could be due to polyploidy effect on maize as earlier observed by .
The control (0.0%) and 0.7% of colchicine treated ACR.91.SUWANI-SRC cultivar shown in plate 1 revealed that the treated plants which produced broader and longer leaves than the control could be as a result of polyploidy induction of the treatment. This supported the findings of [8,9,23], who reported that polyploidy plants differ from there progenitors in terms of broader leaves. The treated plant ACR.91.SUWANI-SRC was found to be resistant to pest attack compared with the control (untreated) which was susceptible to pest in (Plate 2). This could be attributed to the effect of the colchicine treatments as earlier observed and confirmed by the reports of .
Plate 1: Phenotypic expression of two treatment concentrations (0.0% and 0.7%) in ACR.91.SUWANI-SRC. a) ACR.91.SUWANI-SRC. at 0.0% (control); b) ACR.91.SUWANI-SRC at 0.7% colchicine. View Plate 1
Conclusion and Recommendation
Colchicine induced maize genotypes possessed polyploidy characteristics. There was a reduction in percentage germination of seeds as colchicine concentration increased. The colchicine treatment showed a lethal effect and negatively affected the genetic make-up though, enhanced the resistance of maize to pest infestation. ACR.91.SUWANI-SRC genotype tolerated the mutagenic effect of colchicine, could be selected for further genetic improvement of crops.
The authors are immensely grateful to International Institute of Tropical Agriculture (IITA), Ibadan, Nigeria for being the source of the maize genotypes used. Special thanks also go to the Department of Botany, University of Ibadan, Nigeria for providing the necessary facilities and equipment.
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