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ASSESSMENT OF GENETIC DIVERGENCE IN MUTANT LINES OF TOMATO (SOLANUM LYCOPERSICUM L.)

Mutation breeding is the process of exposing seeds to chemicals or radiation in order to generate mutants with desirable traits. This study is aimed at assessing the genetic variability among mutant lines of tomato (Solanum lycopersicum L.) generated from a variety of tomato (Roma VF) using two different chemomutagens. The collected seeds were exposed to different concentrations of Sodium azide and Colchicine with varied period of exposure. The seeds were planted and selected 49 positive mutant lines were assessed for morphological genetic variability and yield. 18 mutant lines that produced fruits were tagged and selected. The fruits of the selected mutants were harvested and the seeds (M2 seeds) were subsequently planted for divergence analysis. Ten quantitative characters and twenty qualitative characters were scored using IPGRI standard tomato descriptor. The potted experiment was laid out in the Green House, using Randomized Block Design (RBD) with three replications. The results of this study revealed a high genetic divergence among the mutant lines in both quantitative and qualitative characters. There was significant LSD (0.05) for Germination percentage (7.66), Plant height at maturity (7.05) and Number of leaves at maturity (4.56). The yield (fresh fruit weight) varied significantly, ranging from 10.00g for LeMT29 to 319.70g for LeMT7 respectively. Fruit and plant qualitative characters equally exhibit variation. These observations suggest the existence of genetic variability among the different mutant tomato lines. Further selection and field trials is recommended to identify suitable and desirable lines for possible variety release. Keywords— Mutant, Tomato, Colchicine, Sodium azide,


INTRODUCTION
Mutation breeding is the process of exposing seeds to chemicals or radiation in order to generate mutants with desirable traits. From 1930-2007 more than 2,540 mutagenic plant varietals have been released that have been derived either as direct mutants (70%) or from their progeny (30%) [1]. Crop plants account for 75% of released mutagenic species with the remaining 25% being ornamentals or decorative plants [2]. In order to speak more clearly about mutations and their potential for crop improvement, it would seem desirable to have different terms at least for (a) the phenotypic alteration and (b) the various underlying molecular and numerical changes. But in any case, a mutation has to be phenotypically expressed to be selectable; all other mutations are only of scientific interest [3]. Mutations are the tools used by the geneticist to study the nature and function of genes which are the building blocks and basis of plant growth and development, thereby producing raw materials for genetic improvement of economic crops [4]. It is a powerful and effective tool in the hands of plant breeders especially for autogamous crops having narrow genetic base [4,5]. During the past 70 years, more than 3,222 mutant cultivars from 175 plant species including ornamentals, cereals, oilseeds, pulses, vegetables, fruits and fibers have been officially released in 50 countries all over the World [1,6,7,8].
The cultivated tomato (Solanum lycopersicum L.) is the most popular garden vegetable and is the second most important vegetable crop in the world in terms of consumption per capital [9,10]. In 2004, tomato assumed the position of one of the most important fruits in terms of Worlds' vegetable produced [11]. Tomato is grown in almost every corner of the planet. On global basis, it is planted 4.6 million hectares of agricultural lands with a total production of 125.5 million metric tons [12]. Nigeria is the 14th largest producer of tomatoes in the world and second only to Egypt in Africa at 1.51 million metric tons valued at N87 billion ($556.1 million) with a cultivated area of 264,430 ha [13]. Both the wet and dry season cropping system contributes immensely to the national requirement. But the bulk production is from the dry season cropping system grown yearly under irrigation system [14]. Tomato belongs to the Solanaceae family, which includes 3,000 species with origins in both the Old (eggplant in China and India) and New World [15].
Several breeders have studied genetic diversity in tomato germplasm for improvement of various growth and yield related traits [16,17,18] but Nigeria has a narrow tomato genetic base, therefore the main aim of this study was to induce variability in a known genotype of tomato (ROMA VF) using two chemical mutagens (Sodium azide and Colchicine) with the specific objectives of selecting positive mutants, evaluation of the selected mutant lines to determine the extent of genetic variability and identification of promising mutant lines for future trials.

MATERIAL AND METHODS
Dried seeds of tomato (var. ROMA VF) collected from the Institute of Agriculture Research and Training (IART) Moore Plantation Ibadan, Oyo state, Nigeria was the foundation seed used in this study. Tomato seeds collected were pre-soaked in water for 24 hours. The presoaked seeds were treated with different concentration of Sodium azide (1.0 × 10-3 mol, 2.5 × 10-3 mol and 5.0 × 10-3 mol) and Colchicine (0.05% and 0.1%) with varied period of exposure (15 min, 30 min and 45 min). The treated seeds were planted in rows having 10-plants per row keeping row-to-row and plant-to-plant distances of 60 cm and 30 cm, respectively with the untreated seeds serving as control.
49 positive mutant lines were identified and tagged as appropriate. The selected 49 positive mutant lines were assessed for morphological genetic variability and yield. The potted experiment was laid out in the greenhouse of the department of Botany, Lagos State University, Ojo Lagos using Randomized Block Design (RBD) with three replications. The blocks contained 10 stands (pots) of each 49 mutant lines, making 30 stands per line (these were the M1 plants). The between and within row spacing of 60cm and 30cm respectively was maintained. 18 mutant lines that produced fruits were tagged and selected. This is represented in Table 1. The fruits of the selected mutants were harvested and the seeds (M2 seeds) were subsequently planted for divergence analysis.
Ten quantitative characters and twenty qualitative characters were scored for using the standard descriptor for tomato [19]. Quantitative data scored for includes; plant height at maturity, number of leaves per plant etc., while qualitative characters include growth habit, stem pubescence etc. Quantitative data collected was subjected to statistical analysis using the Fisher's Least Significance Difference (LSD) test [20].

RESULTS AND DISCUSSION
Considerable variation was observed for all studied traits among the mutant lines. Their phenotypic variations were estimated and shown in different tables and figures. The mean, standard deviation and least significant difference (LSD) of the 10 quantitative characters analyzed for the 18 positive mutant lines that produced fruits are presented in Tables 2. A look at Table 2 reveals that the mean yield (fresh fruit) for the 18 mutant lines (M2) ranged from 10.00g per plant for LeMT29 to 319.70g per plant for LeMT7, the mean plant height at maturity ranged from 46.54cm for LeMT11 to 117.72cm for LeMT49. The average number of nodes at maturity also ranged from 1.20 for LeMT27 and LeMT47 to 5.25in LeMT49. The qualitative characters of Roma VF (control) and the 18 positive mutant lines are presented in Table 3. There was high level of diversity in some characters such as fruit shape, growth habit, stem pubescence, folia density, leaf altitude and degree of leaf dissection. The

LASU Journal of Research and Review in Science
fruit shape of Roma VF (control) is highly rounded. However, the mutagens had effect on the gene(s) controlling fruit shape leading to high level of diversity observed in fruit shape from high rounded in Roma VF to pointed, cylindrical, heart shape, pyriform, slightly flattened and rounded observed in the mutants. The leaf colour, colour of immature fruit and fruit shoulder shape exhibit moderate level of diversity among the mutant lines against the control. While shape of pistil scar, fruit radial cracking and fruit cross-sectional shape exhibited low level of diversity among the mutants against the control.

DISCUSSION
The results of this study indicate a high genetic divergence among the mutant lines against the control in both quantitative and qualitative characters. There was significant difference for germination percentage, plant height and number of leaves among the mutant lines. The yield varied significantly among the positive mutant lines.
The highly significant differences observed using Fisher's least significant difference (LSD) [20] for Germination percentage, Plant height at flowering and number of leaves at flowering among the genotypes evaluated was indication of great deal of variability with respect to these characters. In the same vain, the observed significant variation in the yield among the 18 positive mutant lines also supports the earlier claim that there is a great deal of variability among the genotypes. The observed divergence among the positive mutant lines in reference to both quantitative and qualitative characters was in line with the work of [4] on tomato using Sodium azide and it further confirms that generally, Sodium azide and colchicine are very effective in inducing mutations in tomato. A thorough analysis on quantification of phenotypic variation due to mutagenic effect has also been reported in chickpea [21].
Fruit weight is a quantitatively inherited character that is controlled by many genetic loci; some may have a large effect while others have small effect [22,23,24]. High fruit weights observed may be as a result of the effect of the mutagens on the allele fw 2.2 which influences fruit weight [25]. This allele acts as a regulator of cell division in larger size fruits of tomato [26]. The differences in fruit size observed may be due to the regulators of cell division and cell size acting after anthesis [27].
No significance effect was observed in this population of tomato for number of branches per plant, number of nodes, leaf blade length, and petiole length and as such these characters cannot be used for selection in subsequent improvement program. This observation was consistent with earlier reports by Adamu et al. [28,29] on mutagenic study on groundnut and tomato using gamma rays and that of Sheeba et al. [30] using gammar rays and EMS on Sesanum indicum further confirms that the effects of mutagens on these traits were dose dependent.
There was wide array of variations observed in respect of fruit and plant qualitative characters. The variations were easily recognizable with visual observation. These variations may be as a result of the effect of the mutagens used in this study on the alleles controlling these characters.

CONCLUSION
In conclusion, this study has shown that variation can be easily induced in crop plant using chemical mutagens such as Colchicine and Sodium azide. The level of variation observed after treatment buttress the earlier claim that mutation can raw materials for genetic improvement of economic crops. A number of the positive mutants selected perform better than the original variety (Roma VF).