FACTORS AFFECTING TRANSFORMATION OF GERBERA

1. induction of cell divisions on MS salts supplemented with NaH₂PO₄ vitamins, 10 g sucrose, 0.5 mg/l TDZ and 0.1 mg/l NAA;

2. regeneration of shoots on medium with TDZ lowered to 0.05 mg/l and IAA 0.05 mg/l;

3. growth of separated shoots on ½ MS with 1 mg/l kinetin.
   Petioles of the youngest, still folded leaves taken from proliferating cultures had the best regenerative potential.

Leaf sheets were removed when transferred to regenerative medium.
The first shoots separated after 4–6 weeks were not stable transformed.
Petioles developed organogenic callus which produced single shoots continuously on selective medium.
The first stable transformed shoots were selected from 9-month-old calli.
Under the conditions above 60 – 80% of explants formed shoots with the efficiency from 1 to 6 in two months, depending on the cultivar.
Under the effect of cocultivation with bacteria, both percentage of regenerated explants and number of shoots usually decreased even without selection on kanamycin or PPT. Addition of selective agents to the medium caused further decrease.
Transitional expression of GUS gene was visible on 100% explants and decreased with time, more if kanamycin was not included.
Among three bacterial strains used: EHA 105 containing 35SGUSInt and nptII, EHA 101 containing bar and NPTII, and LBA 4404 containing GUSInt and nptII. The largest number of stable transformed plants was obtained after cocultivation with EHA 101. The criterion adapted for selection was the survival in the course of several passages and proliferation of new shoots on multiplication medium containing 10 mg/l kanamycin or 1 mg/l PPT.