The high frequency of variegated forms after in vitro mutagenesis in Saintpaulia ionantha Wendl.

The leaf-explants of Saintpaulia ionantha Wendl. var.'miniature' were treated by different doses of MNH and cultured on shoot regeneration medium. A strong toxic effect of some MNH doses on explant survival during the first two subcultures was noticed. The explants surviving treatment regenerated shoots with the efficiency comparable to the control. The high number of shoots regenerated from mutagenised leaves showed chlorophyll chimerism (so-called variegated forms). The use of 5 mM MNH for 1.5 or 2 h was found very effective, as 100% of survived explants regenerated variegated shoots. Besides hundreds of variegated forms also leaf-shape and flower-colour variants were observed in MNH-treated culture. Somaclonal variation was not observed in the control culture. The results indicate the great efficiency of in vitro applied MNH for in-duction of morphological variants of Saintpaulia, and especially variegated forms.


INTRODUCTION
African violet (Saintpaulia ionantha Wendl) is one of the most popular ornamental, vegetatively propagated plant and increase of its genetic variation is of economic value. Breed ing of this species has only been achieved by intraspecific hy bridization and sport selection (Grout 1990). Recent develop ment of plant biotechnology can provide new methods for further improvement of Saintpaulia genotypes. Among these new techniques somatic hybridization and genetic transforma tion can be considered. However, these systems require effi cient protoplast culture or transformation system which are not yet developed for Saintpaulia although promissing results on establishment of African violet protoplast culture have been obtained recently (Winkelmann and Grunewaldt 1995;Hoshino et al. 1995). Another way to generate new variation is in vitro mutagenesis (Negrutiu 1990) which can be espe cially effective in plants easily propageted in in vitro culture as African violet. The high morphogenic potential of Saint paulia leaf-explants cultured in vitro has been described (Kukulczanka and Suszyńska 1972) and micropropagation sys tems for this plant have been developed (Bilkely et al 1978;Smith and Norris 1983). The study was carried out to esti mate the efficiency of in vitro mutagenesis in Saintpaulia io nantha leaf-culture in order to induce morphological variants.

MATERIALS AND METHODS
Leaf-explants from plants growing in vitro on the hormone free MS (Murashige and Skoog 1962) medium were used as explants for mutagenesis. Chemical mutagen, MNH, was ap plied in two concentrations: 1 and 5 mM. The mutagen was dissolved in water (1 mM) or in shoot inducing (SI) medium (5 mM) with a few drops of Tween 80. The small leaves with petioles or fragments of bigger leaves (about 1 cm2) were treated on a shaker during 2 or 3 h for 1 mM and 1.5 or 2 h for 5 mM. Following rinsing in water, the treated explants were cultured onto SI medium (MS basal medium; NAA -0.1 mg/1; BA -5.0 mg /I; sucrose -20 g/1; Difco agar -6 g/1; pH 5,8).
After 3 weeks of culture the number of surviving explants was scored and the growing leaves were subcultured onto a fresh SI medium. The procedure was repeated every four weeks. During a total of 8 months, the regenerating shoots were excised and after rooting on RI medium (MS basal me dium; NAA -0.01 mg/1; activated charcoal -6 g/1; sucrose -20 g/1; Difco agar -6 g/1) the plants were planted in the soil.

RESULTS AND DISCUSSION
It was found that all MNH doses decreased the number of explants surviving on SI medium in comparison to the control combination (Table 1). After mutagenic treatment 42.8 to 75.0% of explants displayed growth during the first subcul ture. The toxic effect of the mutagen on African violet leaves and petioles was also observed in the second subculture when the percentage of growing explants in the most harmful com bination (5 mM x 2 h) dropped to 11.4% in comparison to 82.6 % in the control culture. Contrary to the extremely strong effect of MNH on explant survival, no influence of the mutagen on regeneration ability of treated explants was noticed. The regeneration capacity in terms of shoot number developed from surviving explants was very high in all combinations. Shoots differentiation began after 6 weeks in control and after 8 weeks in treated culture from the cut ends of 'explants ( Fig. 1). Shoots were re generated by direct organogenesis as was observed by Ohki (1994). Finally, after 3 months, the entire surface of the ex plant became covered with multiple shoots unabling the cor rect estimation of the shoot number regenerated per explant (Fig. 2). Potentially hundreds of shoots could be excised from one explant but on average 40 to 60 shoots were excised, rooted and transferred to the soil in every combination.
The shoots regenerated from treated explants showed a high level of variation. The highest frequency of variant shoots was induced by the highest MNH doses (5 mM for 1.5 or 2 h) where every treated explant delivered variant shoots ( Table 2). The great prevalence of variants exhibited changes in chloro phyll patterns in leaves, so-called "variegated forms" (Fig. 3). The variegated plants were also noticed after lower MNH doses (1 mM for 2 or 3 h ) but with much lower frequency (up to 6.6 %). Due to "bushy" organogenesis of shoots, the estimation of the number of variegated forms produced per explant was rough. The number of variegated shoots ranged from about 20 to 50% of the total shoot number produced by the treated explant.
The variegated shoots presented a great diversity in chlo rophyll patterns of their leaves. Most of them showed differ ent green-albino sectors (Fig. 4), but plain albino (Fig. 5) and light green plants were also found. Anthocyan variants were also observed but with lower frequency. All these variant types could be observed among regenerants from the same explant. Some mosaic leaves were used as explants. Shoots regenerating from them segregated into full spectrum of chlo rophyll patterns representing both pattern of primary explant as well as new types of variegation or plain plants (albino, light or normal green).
The changes in chlorophyll pattern on leaves of some var iegated plants during their growth were noticed suggesting the presence of heteroplastomatic cells in which sorting-out segregation of plastids resulted in different variegation pat terns (Pohlheim 1981).
Besides the variegated plants, other morphological mutants of African violet, regenerated from different explants were    found with the frequency up to 5%. Variation presented by these plants included leaf shape, so-called serrated leaves (Fig. 6), flower colour (light pink-plain or sectors of albino and pink colour) and plant size. It should be stressed that all variants were found only in mutagenised culture. Contrary to Jain (1993) suggesting that BA can be responsible for vari ation in Saintpaulia ionantha plants regenerated in leaf-disc culture, in the presented experiment non somaclonal variants were observed in the control culture. The genetic determination of induced variants is not proved but the high frequency of variegated and other forms found only after mutagenic treatment indicates their genetic back ground. Considering MNH as a very efficient plastome mut agen (Hagemann 1982) and suggestion of Pohlheim and Ber ger (1974), cytoplasmatic determination of variegated forms can be assumed.
It was indicated that from one MNH-treated leaf-explant dozen of variant plants could be obtained. The results showed the great efficiency of in vitro mutagenesis in Saintpaulia io nantha and advantages of this method over mutagenesis of in vivo formed adventitious buds of African violet (Warfield 1973;Pohlheim 1974).