Organogenesis and long-term micropropagation of Polish pea cultivars

Abstract

The complete protocol for regeneration and long-term micropropagation of several Polish cultivars of pea (Pisum sativum L.) has been elaborated. The shoots were the most likely regenerated via de novo organogenesis. The adventitious buds formed in callus derived from cotyledons tissue adjacent to the axillary meristems of immature embryos. All cultivars' calli regenerated several shoots per explant on the MS medium supplemented with B5 vitamins and 4.5 mgl-1 of BAP, however some differences in regeneration capacity among cultivars were observed. The plantlets were subsequently micropropagated with slightly higher efficiency and preserving a good viability over the long-term culture on a medium containing 2.0 mgl-1 than one with 4.5 mgl-1 of BAP. The additional step of the pre-conditioning culture of multiplicated shoots on a medium with very low BAP concentration i.e. 0.02 mgl-1 was applied and appeared to be beneficial before rooting in vitro or grafting. The modified MS-derived medium with the half-strength of MS macroelements but with the full original dose of calcium and supplemented with B5 vitamins and 1.0 mgl-1 of NAA was developed for effective rooting. The shoots were also sufficiently transferred into ex vitro conditions using grafting. The majority of the regenerated plants had adapted to in vivo conditions in a greenhouse and subsequently has set seeds. The presented protocol provides relatively efficient rate of de novo pea regeneration and would be useful for Agrobacterium-mediated transformation purposes.