Enhancement of somatic embryo yield from carrot cell cultures by extracellular factors

An important factor in the development of successful, commercial plant propagation through somatic embryogenesis is the conversion yield of cells to somatic embryos. This study explored the growth factor-like potential of high molecular weight, extracellular compounds to elucidate and accelerate the developmental process of carrot somatic embryogenesis.; When spent culture medium extracted from embryo cultures grown in the absence of 2,4-dichlorophenoxy acetic acid (2,4-D), referred as embryo-free medium (EFM), was added to suspension culture of carrot, large and rapid increases in embryo production were observed. In order to further investigate the responsible cell factors, EFM was concentrated into several M.W. sizes by ultrafiltration membranes to determine which size of in vitro compounds, what optimum concentration of EFM, and what culture period harvesting time for EFM can stimulate embryogenesis. Highest embryo production was obtained with the addition of concentrated EFM ({dollar}>{dollar}50 kDa) containing 2.8 {dollar}mu{dollar}g protein/ml. High molecular weight proteins larger than 50 kDa and a late exponential phase harvest of EFM showed the highest potential to increase the production of end stage torpedo embryos as well as plantlets. Enhancements of 2 to 3 times compared with that of control cultures in a batch process could be obtained. Salt precipitation and heat treatment of EFM suggest that the causative agent may be excreted protein molecules. Such proteins could function as growth factors related to differentiation and embryo production. The same enhancing effect found in a shake flask was observed in an airlift fermentor after the addition of EFM protein factors. From SDS-polyacrylamide gel electrophoresis (PAGE) experiments, 72, 82 and 95 kDa proteins were found as unique embryo-specific polypeptides which have the capability to stimulate carrot somatic embryogenesis.