RESUMO
The aim of this study was to check the effects of sugar type on the in vitro shoot multiplication of the tulip cultivar 'Heart of Warsaw' and the effects of paclobutrazol (PBZ) and 1-naphthylacetic acid (NAA) on the bulbing of previously multiplied shoots. In addition, the subsequent effects of previously used sugars on the in vitro bulb formation of this cultivar were checked. First, the optimum supplementation of Murashige and Skoog medium with plant growth regulators (PGRs) was selected for shoot multiplication. Of the six tested, the best results were obtained using a combination of 2iP 0.1 mg·L-1, NAA 0.1 mg·L-1, and mT 5.0 mg·L-1. The effects of different carbohydrates (sucrose, glucose, and fructose at 30 g·L-1 and a mixture of glucose and fructose at 15 g·L-1 each) on multiplication efficiency was then tested on this medium. The microbulb-forming experiment was carried out taking into consideration the effects of previously applied sugars, and at week 6, the agar medium was flooded with liquid medium containing NAA 2 mg·L-1, PBZ 1 mg·L-1, or medium without PGRs; in the first combination, the cultures were left on a single-phase medium, solidified with agar, as a control. After 2 months of treatment at 5 °C, the total number of microbulbs formed and the number and weights of mature microbulbs were assessed. The results obtained indicate the ability of using meta-topolin (mT) in tulip micropropagation and point to sucrose and glucose as the optimal carbohydrates for intensive shoot multiplication. The results lead to the conclusion that it is most advantageous to multiply tulip shoots on glucose medium and then to carry out cultures on a two-phase medium with PBZ, which results in a higher number of microbulbs and their faster maturation.
RESUMO
We describe here a new tulip micropropagation method based on the cyclic shoot multiplication in presence of the thidiazuron (TDZ), which enables the production of virus-free stock plants, speeds up breeding, and provides new genotypes for the market. In our novel protocol, cyclic shoot multiplication can be performed for 2-3 years by using TDZ instead of other cytokinins, as 6-benzylaminopurine (BAP) and N(6)-(-isopentyl)adenine (2iP). It makes possible to produce 500-2,000 microbulbs from one healthy plant. There are six main stages of tulip micropropagation. Stage 0 is the selection of true-to-type and virus-free plants, confirmed by ELISA. Fragments of flower stems isolated from bulbs are used as initial explants. Shoot multiplication is based on the regeneration of adventitious shoots, which are sub-cultured every 8 weeks. In the Stage 3, the specially prepared shoots are induced by low temperature treatment to form bulbs which finally develop on a sucrose-rich medium at 20 degrees C. Bulbs are then dried for 6 weeks and rooted in vivo. The number of multiplication subcultures should be limited to 5-10 cycles in order to lower the risk of mutation. Virus indexing should be repeated 3-4 times, at the initial stage and then during shoot multiplication. Genetic stability of micropropagated shoots can be confirmed using molecular markers.
