THE INFLUENCE OF THE MINERAL COMPOSITION OF THE MEDIUM ON IN VITRO PROPAGATION OF KOHLERIA AMABILIS (PLANCH. ET LINDEN) FRITSCH SHOOTS

The aim of the study was to test the influence of the mine ral salt composition of the medium on in vitro multiplication and growth of kohleria shoots. Shoot tips were cultured on Murashi ge and Skoog (MS) (full and half strength), Gamborg et al. (B5), Nitsch and Nitsch (NN) or Lloyd and McCown (WPM) media supplemented with BA 1 mg·dm 3. The influence of passa ge time on the induction and growth of shoots on the medium con taining half strength of MS mineral salts and BA 1 mg·dm 3, GA3 1 mg·dm 3 was also studied. The explants used in the experiments were obtained from aseptically grown shoot clusters. A signifi cant influence of the medium type and the time of propagation on the number, length and weight of axillary shoots was observed. The medium containing full or half strength of mineral salts ac cording to MS was the best. The propagation of kohleria shoots should take place at 4 week passages.


INTRODUCTION
In horticultural production, the traditional methods of propagation of ornamental plants are more and more frequently replaced by the in vitro culture method. This technology allows healthy and homogenous material to be obtained for sale in a short time. It is of special significance in the case of valuable plant species, but which are little known on the floriculture market. In available literature, there are reports on micropropagation of many plants from the family Gesneriaceae  An appropriately selected medium has an essential influence on the morphogenesis and the achievement of desired effects in tissue cultures. The effect of growth regulators on explants growing in vitro is possible thanks to supplying plants with essential nutrients, notably mineral salts. There are many types of growth media with a strictly defined mineral composition, e.g.  1980). The multiplication stage is composed of passages the time of duration of which is adapted to the respective plant species and variety in order to obtain the highest possible multiplication ratio. During the duration of the passage, the chemical composition of the medium changes and adversely affects plant quality.
The aim of this study was to test the influence of the mineral composition and the time of passage on the induction and growth of kohleria shoots.

MATERIAL AND METHODS
Shoot tips and nodal sections of shoots taken from kohleria plants growing in a greenhouse were used as explants in the experiments. They were sterilized in sodium hypochlorite solution containing 0.75% of active chlorine for 30 min. and rinsed three times in sterile water. The explants were placed on modified N i t s c h and N i t s c h (NN) (1969) medium supplemented with kinetin (1mg·dm -3 ) and GA 3 (1 mg·dm -3 ). Regenerating shoots were multiplied in 4-week passages on medium containing 1 / 2 macro-and micronutrients according to M u r a s h i g e and S k o o g (MS) (1962), NaFeED-TA 40,3 mg·dm -3 and organic compounds: 0.4 mg·dm -3 thiamine, 0.5 mg·dm -3 pyridoxine, 0.5 mg·dm -3 nicotinic acid, 2 mg·dm -3 glycine, 100 mg·dm -3 myo-inositol, 30 g·dm -3 sucrose, BA 1 mg·dm -3 , GA 3 1 mg·dm -3 . After several passages of multiplication, shoot tips of 6-10 mm in length, with 2 pairs of well developed leaves, were dissected from the shoot clusters and used in the experiments.
In the first experiment, the explants were cultured on the media containing mineral salts according to:  (1980). Every medium contained iron and organic compounds as at the initial stage and BA 1 mg·dm -3 . In the second experiment, medium contained half strength of MS mineral salts, iron, organic compounds as at the initial stage and growth regulators: BA (1 mg·dm -3 ), GA 3 (1 mg·dm -3 ). All the media were solidified with 6.5 g·dm -3 Agar-Agar (Sigma). The pH of the media was adjusted to 5.7 before autoclaving.
Seven shoots were incubated per 250 ml Erlenmeyer flask. Twenty one shoots were used for each combination. Each flask with 7 explants was a replicate. The experiment was repeated twice.
The cultures were maintained at 22°C with a photon flux density of 35 µM . m -2. s -1 and a 16-h photoperiod. The first experiment lasted 4 weeks. In the second experiment, subculturing was performed after 3, 4, 5 and 6 weeks. The number of axillary shoots from 1 explant, their length and fresh weight, and fresh weight of shoot clusters were determined. The results of the experiment were analyzed statistically using a standard statistical procedure with one factorial design and the Tukey test was used to estimate the differences between the means at a 5% level of significance.

RESULTS AND DISCUSSION
Analysis of the study results showed a significant influence of the medium type on the number of shoots formed from an explant. The largest number of shoots was obtained on 1 / 2 MS medium and standard MS medium, whereas the lowest number on NN and B 5 medium (Tab. 1). WPM medium had a smaller effect on the trait studied.
It was found that the shoots growing on NN and B 5 medium formed the significantly longest axillary shoots. The significantly shorter shoots were obtained on WPM and 1 / 2 MS medium. In turn, MS medium had a slightly weaker stimulating influence on shoot growth than WPM and 1 / 2 MS medium.
Among the medium types studied, B 5 showed the significantly highest influence on the fresh weight of axillary shoots. Shoots with the significantly lowest fresh weight were obtained on NN and WPM medium. The favourable effect of 1 / 2 MS and MS medium on the abovementioned trait was also observed.
A significant influence of the medium type on the fresh weight of the shoot cluster was demonstrated. Shoot clusters obtained on 1 / 2 MS and MS medium were characterised by the highest weight, whereas the lowest weight was noted on WPM medium. NN and B 5 medium indirectly influenced the trait studied.
A significant influence of the time of passage on the growth and development of kohleria shoots was also noted. The largest number of axillary shoots was obtained during the passage lasting 6 weeks (Tab. 2). These shoots were also characterised by the largest length and the highest fresh weight compared to the shoots origi-  nating from the 3-week passage. Over the same period, a shoot cluster with the significantly highest fresh weigh was obtained. At the same time, it was observed that the quality of the new shoots worsened as the passage was prolonged. They became excessively elongated and had smaller leaves. Apart from that, a large part of the leaves browned. The shoots after 4 weeks of the duration of in vitro culture showed the best quality. The chemical composition of the medium is one of the factors determining the success of in vitro cultures (K ę p c z y ń s k a and K ę p c z y ń s k i , 1998). In this study, it was shown that the mineral composition has a significant influence on the induction and growth of kohleria shoots. Based on the results, ½ MS and MS media were singled out as the ones which have the most favourable effect on regenerating kohleria shoots. The duration of the passage during the multiplication of kohleria had a significant influence on the traits studied. During the 6-week passage, the highest multiplication ratio was obtained, but the quality of the new shoots was unsatisfactory.