Abstract
BACKGROUND AND AIMS: Microspore embryogenesis is a convenient inducible system to study the changes associated with the developmental reprogramming of cells. In this work, Brassica napus microspore cultures were used to study the role in the embryogenic switch of callose and pectin cell wall composition, which depends on Ca2+ levels. METHODS: We used different chemicals to modulate Ca2+, callose and pectin methyl-esterification, including Ca(NO3)2, inositol 1,4,5-trisphosphate, 2-deoxy-d-glucose, benzyl alcohol, chitosan, epigallocatechin gallate and pectin methyl-esterase. Ca2+ distribution, callose and cellulose were imaged with FluoForte, Aniline Blue and Pontamine Fast Scarlet stainings, respectively, and observed with confocal microscopy. KEY RESULTS: Inhibition of callose synthesis with 2-deoxy-d-glucose demonstrated that callose is essential for induction of microspore embryogenesis. A moderate increase of Ca2+ levels with Ca(NO3)2 or inositol 1,4,5-trisphosphate promoted increased callose synthesis and deposition in the cell wall. However, the use of benzyl alcohol and chitosan to permeabilize the plasma membrane and allow for Ca2+ influx was not positive, because this prevented embryo development by inducing callus formation. Benzyl alcohol did not affect callose and cellulose deposition, but chitosan induced the formation of callose plugs, similar to those formed in response to pathogen attack. Inhibition of pectin methyl-esterase activity with epigallocatechin gallate during the first 3 days of culture produced ~70 % more embryos, but prolonged exposures were negative. Instead, increased pectin methyl-esterase activity during the first 3 days was not positive, but when applied for 7 days, embryos increased by ~60 %. CONCLUSIONS: Together, these results confirm the relevant role of calcium and callose during the first stages of microspore induction and suggest that the levels of pectin methyl-esterification in the cell wall are dynamic and that different cell wall compositions are required during the different stages of microspore embryogenesis.