Callus induction and growth in transgenic potato genotypes

The aim of this study was to establish an effective protocol for callus induction from the potato genotypes ( Solanum tuberosum L.) used and to investigate whether the transferred oxalate oxidase enzyme or transformation procedure has any effect on callus induction of transgenic lines of cultivar Desiree and Maris Bard. The results showed that the effects of genotype and medium on measured characters were highly significant. In order to compare overall performance of transgenic lines of each cultivar with their parental cultivars (non-transgenic), orthogonal comparisons were used and indicated the significant differences between transgenic and non-transgenic genotypes. The significant differences between the cultivars and their transgenic lines expressing oxalate oxidase enzyme indicated that the transformation procedure had a significant effect on their callus induction and growth.


INTRODUCTION
Much work has been carried out on callus induction and growth in potatoes (Solanum tuberosum L.).This has resulted in a range of protocols and procedures being established by researchers since tissue culture gained an importance in plant propagation, conservation and breeding (Ahloowalia 1982;Wareh et al., 1989).Previous researches showed that media used for callus induction and growth depends on the genotypes Gonzalez et al. (2001, Alexeenko andIrkaeva (1998) pointed out that introduction of genes effecting the structure and type of plant development into strawberry lines also influenced callus formation and shoot inducing in vitro.This depicts involvement of inheritance in callus growth.In the last two decades, many transgenic plants have been produced by using a range of transformation methods.
Callus is used for most of these transformation methods such as particle gun (McCabe et al., 1998) and Agrobacterium tumefaciens-mediated transformation (Stiekema et al., 1988) as well as initiation of cell culture.A callus from an explant tissue occurs as a result of dramatic changes in the appearance and metabolism of the cells (Aitchison et al., 1978).Induction of callus, physical disorganization of cultured cells, is thought as result of the breakdown of intercellular physical and chemical communication (Lindsey and Jones, 1992).In this transportation system, vascular bundles in a plant play important roles in terms of transferring water and plant nutrients.Recently, some enzymes such as oxalate oxidase, which is more expressed in salt tolerant species, are thought to have an effect on the vascular system (Hurkman and Tanaka, 1996).
Therefore, the aim of this study was to establish an effective protocol for callus induction from the potato genotypes.Secondly, it was to investigate whether the genetic transformation procedure and oxalate oxidase transgene have any effect on callus induction in potato genotypes.

MATERIALS AND METHODS
Two potato cultivars (Maris Bard and Desire) were obtained from the collections of Department of Agricultural Botany at The University of Reading, United Kingdom.Three transformed genotypes (MB T1 , MB T2 and MB T3 ) were derived from cultivar Maris Bard (MB), and one (DS T1 ) derived from cultivar Desiree by using A. tumefaciens-mediated transformation method (Turhan, 1997).Transformed genotypes expressing oxalate oxidase enzyme were initially conformed by using kanamycin resistance as the selective marker and the oxalate oxsidase enzyme activity as the reporter gene support the expression of transgene.All genotypes were grown on Murashige and Skoog (1962) basal culture medium without any growth regulators.
Several plant parts such as leaf, root, stem and tuber have been used successfully for callus induction in potato (Ahloowalia, 1982;Austin, 1989;Osifa, 1989).In our case, stem segments were chosen as primary studies showed that stem section was the most responsive explant for the cultivars.
The stem segments, approximately 0.5 cm long and uniform diameter (approximately 1 mm), were excised from four week old in vitro plantlets.Excised stems without axillary buds, for each genotype, were collected in a Petri dish containing sterile water to randomise the explants before transferring to the media.
The major growth regulators that have been used successfully in callus induction and growth are kinetin, 2, 4-dichlorophenoxy acetic acid (2,4-D), α-naphthaleneacetic acid (NAA), indole-3-acetic acid (IAA), zeatin and gibberellic acid (Osifa,1989;Szabo et al., 1994).Therefore, three different modified callus induction media based on the basal MS medium and vitamins were used (Table 1).After preparation of the media, Petri dishes were divided into two equal parts by drawing a middle line.Each half of the Petri dishes was inoculated with four stem segments.
The number of shoots and roots: Shoots and roots arising from a single explant were counted (if any).This character was not subjected to statistical analysis as few explants had shoots and roots and it is also not desired in callus culture.
The experimental design was a randomised block factorial design with six replicates.Analyses were carried out for these characters on transformed data.The statistical analysis was carried out by using the SAS computer package (SAS Inst. 1998).

RESULTS AND DISCUSSION
In order to evaluate effect of oxalate oxidase enzyme on callus induction and growth, the results were recorded for stem segments from the transgenic and non-transgenic potato genotypes growing on a basal MS medium supplemented with three different combinations of growth regulators.The data were analysed and the analysis showed that the effect of genotype and medium on characters measured was found to be highly significant (Table 2).
In order to compare overall performance of transgenic lines of each cultivar with their parental cultivars (nontransgenic), orthogonal comparisons were used and indicated the significant differences between transgenic and non-transgenic in terms of some characters measured (Table 2).In media M2 and M3, none of calli had shoots whereas few shoots were observed in M1.However, the root and shoot data were not included since our first aim was not regeneration.In addition, there were no roots in M1 and when the cultures were kept more than four weeks.Few and short roots occurred in M2 and M3.
When the media were compared in terms of overall scores of all genotypes, the calli on M2 were bigger and had growth better than those on M1 and M2.At the same time, in terms of callus quality it varied from 'intermediate' to 'friable' in type and produced roots (Although the roots were not very long, and less than 1 cm).On the other hand, the calli on M1 tended to produce shoots.After sub-culturing of calli on M2 for bulking purposes, a dramatic reduction was observed in the number of roots.As a result, the best medium for callus induction was M2 (Table 3) The significant differences between transgenic and non-transgenic (parent) genotypes indicate that transformation procedure had an effect on callus induction.A number of research projects have indicated the relationship between the oxalate oxidase activity of  germin and osmotic and salinity stress in barley and wheat plants (Hurkman et al., 1991; Hurkman, 1992; Lane et al., 1993;   Lane, 1994; Hurkman and Tanaka, 1996).At the same time, Zhang et al. (1995) pointed out that oxalate oxidase enzyme in barley degrades oxalate produced in fungal infected sites of plant tissue.The degradation process of oxalate gives H 2 O 2 and CO 2 (Thompson et al., 1995).Similarly, Ca 2+ and H 2 O 2 can be released by degradation of calcium oxalate by oxalate oxidase which is in extracellular matrix (Lane, 1994).It can be speculated that the enzyme itself and its products or both may play important roles in the cell wall matrix in terms of water transport (Campbell and Sederoff, 1996), ion selectivity (Showalter, 1993) and cell wall lignification (Halliwell, 1978;Goldberg et al., 1987) by cross linking reaction in higher plants.Moreover, Lindsey and Jones (1992) stated that induction of callus or physical disorganization of cultured cells is thought as a result of the breakdown of intercellular physical and chemical communication.Therefore, the presence of oxalate dase In potato genotypes might have increased communication of the cells within a callus.Consequently, callus induction and growth in transgenic lines decreased when compared to calli of non-transgenic genotypes (Table 3).
In conclusion, the differences between the cultivars and their transgenic lines in terms of callus growth indicated that transformation procedure or the oxalate oxidase enzyme had a role in this complex mechanism.However, further investigation is required to determine whether this effect comes from transformation procedure alone or oxalate oxidase enzyme.

Table 1 .
Formulation of three media used for callus induction and growth.

Table 2 .
Analysis of variance for characters measured (mean square).

Table 3 .
Effect of different media on callus induction and growth in potato genotypes.Means with the same letter in a line are not significantly different at p=0.05. *: