Professional paper
Micropropagation of White Poplar (Populus alba L.)
Bojana Pintarić
; Sarajevo
Abstract
The plant material used in this experiment derives from the laboratory Firenza Scienza e Tecnologia Ambientali Forestali. It had already been introduced into the culture so that in this paper we only conducted the procedures of shoot multiplication, shoot lengthening, shoot restoration and their acclimatization. Namely, the plants were taken from Erlenmeyer flasks in sterile (aseptic) conditions. The damaged leaves and the callus were removed with a scalpel. The tree was then segmented into individual explants with at least one node of 1–2 cm. The dissected explants were transferred to the fresh multiplication medium. The culture was multiplied in the basic medium: modified Woody Plant Medium (WPM; MS /Murashige and Skoog/ - macroelements, WPM – microelements and vitamins) with the addition of 0.25 or 0.5 mg/l BA (6-benzyladenine).
The explants were transferred to fresh mediums every 28–35 days, which in fact represented the subcultivation period (passaging) during the nine-month duration of the experiment (from November 2006 to June 2007). The growth of the explants was slowed or halted due to impoverished nutrients, the drying of the agar, the production of poisonous bioproducts and lower quantity of oxygen in the flask. Therefore, only the healthy tissue was subcultivated. The shoots were multiplied in multiplication mediums; in other words, new shoots were differentiated by induction from lateral buds. Approximately one month later new lateral branches could easily be identified and removed from the parent tree. At this period it was noticed that the shoots behaved differently in the mentioned mediums. In order to assess potential multiplication and the multiplication rate of a given species, the potential explants were separated from the parent explant and transferred to the new medium.
The shoot multiplication rate in these mediums varied and amounted to 5.36 per explant for M1 medium and to 5.86 for M2 medium during the first five passages. However, in the subsequent passages the lines were clearly separated and the number of new shoots per explant on M1 medium dropped to the average 1.8; whereas it rose to 13.45 per explant on M2 medium.
The growth rate of the plant material of white poplar under in vitro conditions was proportionate to the multiplication rate in both types of multiplication mediums. However, plant growth rate in M1 medium was more distinct compared to M2 medium, where this relationship changed very quickly and clearly in the three following passages. Thus, the M2 medium proved superior for long-lasting use of these explants under in vitro cultures.
A part of the explants was “sacrificed” at different time intervals in order to obtain and measure mean fresh and dry mass values. After “sacrificing” the explants and collecting fresh and dry mass data, some basic statistical data were calculated (Table 4) for the growth rate of the plant material under in vitro conditions. According to the data, the shoots cultivated in the M1 medium had mean fresh mass values of 0.04022 g and dry mass of 0.00888 g. The subsequent control measurements showed the mean fresh mass value of 0.03588 g and dry mass value of 0.00630 g. On the other hand, the explants cultivated in the M2 medium showed fresh mass values of 0.03851 g and 0.00743 g and dry mass values of 0.02988 g and 0.01397 g.
Isolated shoots sized about 3-4 cm showed 100 % rooting under in vitro conditions after being planted into the rooting medium (Z) with the addition of 0.1 mg/l IBA (Indol Butric Acid) for seven days and their transplanting into the basic hormone-free medium (BH) in the following tree weeks. In the acclimatization stage, the rooted plants were transferred from the sterile containers in which they were cultivated into the soil substrate (soil and sand at 3:1 ratio) outside the chamber climate.
The root system was cleaned from the medium particles with agar using a spray of sterile distilled water. The plants were transplanted into the soil substrate previously sterilized with fungicide. These young plants did not have a sufficiently developed cuticula, a functional stomatal apparatus, a good vascular connection between the root and the shoot, well developed root hairs, etc., so it was necessary to acclimatize them gradually to the ex vivo conditions. This was achieved with temporary protection of the young plants with sterile glass containers. The removal of the containers was strictly controlled and the period of removal was lengthened every day in order to enable gas exchange. On the other hand, for the first seven days the plants were watered with sterile distilled water and after that with tap water.
Although this is usually a critical stage, all the transplanted individuals managed to acclimatize to relatively low air humidity prevailing in external conditions (ex vitro conditions). No phenotypal changes were observed on the plant material either in in vitro or in vivo conditions during the experiment.
Keywords
Hrčak ID:
26354
URI
Publication date:
31.8.2008.
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