Applied stress is often used to induce embryogenesis in microspore culture. Cold pretreatment has been used in cereal crops, Brassica napus and Brassica rapa microspore cultures, but seldom attempted in Brassica oleracea microspore culture. In this study, the effect of cold pretreatment (4°C) on embryogenesis in microspore culture and ploidy of regenerants resulting from those cultures was investigated in three broccoli cultivars ÔGreen EliteÕ, ÔGreen StarÕ and ÔTI-111Õ. Cold pretreatment for 1, 2 or 3 days was effective when applied alone using these three cultivars, but the frequency of embryos derived from microspores was low. The combination of cold pretreatment (4°C) for 1 or 2 days and heat shock (32.5°C) for 1 day significantly enhanced microspore embryogenesis efficiency, especially with ÔTI-111Õ for which it was increased by 63-72-fold. In addition, the combined treatment resulted in higher diploid frequency of the regenerated population compared to traditional microspore culture protocol which typical only uses heat shock at 32.5°C for 1 day in broccoli.

SummaryThe nature of pathogenic mechanisms associated with the development of multiple sclerosis (MS) have long been debated. However, limited research was conducted to define the interplay between infiltrating lymphocytes and resident cells of the central nervous system (CNS). Data presented in this report describe a novel role for astrocyte-mediated alterations to myelin oligodendrocyte glycoprotein (MOG)35-55-specific lymphocyte responses, elicited during the development of experimental autoimmune encephalitomyelitis (EAE). In-vitro studies demonstrated that astrocytes inhibited the proliferation and interferon (IFN)-g, interleukin (IL)-4, IL-17 and transforming growth factor (TGF)-b secretion levels of MOG35-55-specific lymphocytes, an effect that could be ameliorated by astrocyte IL-27 neutralization. However, when astrocytes were pretreated with IFN-g, they could promote the proliferation and secretion levels of MOG35-55-specific lymphocytes, coinciding with apparent expression of major histocompatibility complex (MHC)-II on astrocytes themselves. Quantitative polymerase chain reaction (qPCR) demonstrated that production of IL-27 in the spinal cord was at its highest during the initial phases. Conversely, production of IFN-g in the spinal cord was highest during the peak phase. Quantitative analysis of MHC-II expression in the spinal cord showed that there was a positive correlation between MHC-II expression and IFN-g production. In addition, astrocyte MHC-II expression levels correlated positively with IFN-g production in the spinal cord. These findings suggested that astrocytes might function as both inhibitors and promoters of EAE. Astrocytes prevented MOG35-55-specific lymphocyte function by secreting IL-27 during the initial phases of EAE. Then, in the presence of higher IFN-g levels in the spinal cord, astrocytes were converted into antigen-presenting cells. This conversion might promote the progression of pathological damage and result in a peak of EAE severity.