The Protein synthesis spectrum during the induction phase of somatic embryogenesis in Carrot (Daucus carota L.) cultures and the role of Nitrogen forms for embryo development

Abstract

The goal of the investigation was to broaden our knowledge about the pattern of protein synthesis during the induction phase of somaticembryogenesis in Daucus cultures using petioles as the source material implementing histological, bio-chemical, molecular andbio-informatics methods to achieve this aim for a better understanding of the somatic embryo-genesis in the model system carrot. Duringthe induction phase, histological examination of cross sections of petiole ex-plants of Daucus carota in Gamborg medium with aconcentration of 0.5 ppm 2,4-D liquid media shows three different zo-nes, namely, a rhizogenic area with the appearance of cytoplasm richcells and first cell division located around the vascu-lar bundles 2 days after culture initiation in an auxin containing medium (Schäfer et al.,1988). Root primordia appear 5 days after culture, and at 7 days of the culture adventitious roots appear. Second, a caulogenic area whicharises from the parenchyma, with first cell divisions 5 days after culture in an auxin containing medium. Shoot primordia appear 12 daysafter culture, and finally embryogenic areas appear which are characterized by cytoplasm rich cells and first cell divisions in subepidermalregions 12 days after culture in auxin containing media. These cytoplasm rich embryogenic cells, when transferred to an auxin free medium,divide and give rise to globular structures which proceed to torpedo and finally mature embryo forms. This trend can be seen throughstaining the petiole transection with hematoxylin. To study the pattern of the protein synthesis, the cultured petiole explants were labelledwith 14C-Leucine. Comparison of the histora-diogram of t0 (t5h), t7 and t14 section indicates a preferential accumulation of 14C-Leucine inthe active morphogenic areas at different stages of culture. Coomasse brilliant blue R-250 stained and 14C-Leucine labelled 2-DE ofprotein spots in a 5 hours, 7 days and 14 days old carrot petiole culture showed 91, 250 and 256 spots respectivly (Grieb et al., 1977). 71spots appeared at all periods, however, each period had its own specific proteins. Some spots were merely stained, some merely labelled,and some stained and labelled. Global protein analysis is a procedure to identify proteins and their func-tional analysis using anidentification data base, in this case Swiss Prot, through subtractive analysis and comparison on the basis of protein spots variation,additional spots, missing spots, using MW and pI of the protein spots for identificati-on. To examine the role of nitrogen, particularly duringthe realization phase of somatic embryogenesis in carrot petiole and suspension cultures and the role of pH, inorganic reduced andoxidized forms such as (NH4)2 SO4 and KNO3 and an organic form of nitrogen in form of casein hydrolysate were used. According to theresults obtained, ammonium sul-fate as the reduced form of nitrogen in higher concentrations reduces pH of the liquid medium to a range of4.00 on the pH scale, which leads to the arrest of embryo development. By using potassium nitrate, globular structures developed intoheart, torpedo, mature embryos, and finally plantlets were formed. Potassium nitrate increased the pH of the liquid medi-um to 7.2. Usingcasein hydrolysate at the same nitrogen concentration as the inorganic nitrogen source, embryo deve-lopment was arrested at the latetorpedo stage. This form of nitrogen, having a close relationship between its pH and pK, acts as a buffer, therefore, pH of the liquid solutionmedium was not strongly subjected to variation. A very important character of the selected nitrogen form used is their effect on the pH of theculture medium. A mixture of three different nitrogen forms in the solution had a pH capacity between the organic and inorganic nitrogencontaining solution. So the solution makes use of different characteristics of each nitrogen form. Diammonium sulfate reduces pH of thesolution, potassium nitrate exerts an opposite effect by increasing pH, and finally casein hydrolysate acts as buffer to stabilize the pH of thesystem.