| Agapanthus praecox ssp. orientalis are evergreen and semi-evergreenherbaceous perennial flowers, originated in southern Africa. As tropicalplant, due to the high water content of cells, poor cold tolerancecharacteristics, Agapanthus embryonic callus showed are more sensitive tostress responses during cryopreservation process by vitrification stress.Plant cryopreservation based on vitrification has been applied veryextensive, relatively few studies on plant stress physiology responsemechanisms during the cryopreservation process. This paper studied thecellular ultrastructure, cell membrane qualitative composition, Ca2+distribution and comparative transcriptomics agapanthus of embryoniccallus tissue at different cryopreservation key steps to explore Agapanthusembryogenic callus stress response mechanism during cryopreservationprocess to provide the further optimization for Agapanthuscryopreservation systems theory and reference for the cryopreservation ofother plants, concrete results are as follows:1. Agapanthus embryogenic callus cell ultrastructure was observedduring cryopreservation process, cell wall and cytoplasm separated,membranous structure intact after preculture; cell wall separating thecytoplasm, protoplast concentrated, organelles were damaged, moreintracellular vesicles appeard after dehydration; after cryopreserved inliquid nitrogen then recovery, the separation of cell wall and cytoplasmrecovered partly, intracellular mitochondria structure integrity, the stategradually returned to normal cells, some cell structure and cell inclusionscollapsed. Description reversible structural changes are an adaptive response of cells to protect themselves against environmental stress, andirreversible damage caused cell death.2. Agapanthus embryogenic callus were detected two kinds ofunsaturated fatty acids, which are oleic acid (C18:1), linoleic acid (C18:2);five saturated fatty acids, which are palmitic acid (C16:0), stearic acid(C18:0), arachidic acid (C20:0), behenic acid (C22:0) and lignoceric acid(C24:0)，with GC/MS method. The palmitic acid is the highest contentsaturated fatty acids. The control palmitic acid content was31.41%, thecontent of the preculture increased to36.11%, after dehydration reduced to35.31%, the content of the recovery increased to the maximum of44.27%.So speculated that palmitic acid induced mitochondrial dysfunction leadingto cell death. Unsaturated fatty acids in the cell culture after preculture wasreduced to30.23%, after dehydration treatment increased to a maximum of35.91%. The change of linoleic acid (C18:2) content was the mostsignificant, indicating Agapanthus EC mainly by increasingpolyunsaturated fatty acids resisted cold stress. Elevated levels ofunsaturated fatty acids help maintain fluidity and stability of cellmembrane, and reduce environmental stress damage to the cell membrane.3. Four key steps of Cryopreservation by vitrification, preculture,loading, dehydration and dilution on changes of Ca2+distribution inAgapanthus praecox embryogenic callus were investigated by antimoniteprecipitation method and fluorescent probe Fluo-3AM staining. The resultsshowed that calcium antimonite precipitates represented Ca2+distribution,after perculture, vacuole contained more amounts of Ca2+than thecytoplasmic matrix, most of Ca2+cling to the membrane, the cellfluorescence intensity declined to the minimum19.36%. After loadingtreatment, many starch granules and vesicles appeared in cells, calciumantimonite precipitates were observed in mitochondria and vesicles, thenumber of calcium antimonite precipitates and cell fluorescence intensityincreased to24.56%. After dehydration treatment, cell fluorescenceintensity rose to the maximum36.74%and maintain gradient, the number of vesicles containing Ca2+increased, large amounts Ca2+of aroundintracellular starch grains. After dilution treatment, the number ofintracellular Ca2+and the fluorescence intensity decreased to32.95%,cytoplasmic Ca2+concentration changed back to normal (ck) levels with theintracellular vitrification solution reduced. The number of starch grainsincreased but less Ca2+distributed around starch grains. It was deduced thatchanges of Ca2+determined different adaptability for the composite stressduring cryopreservation.4.Agapanthus embryonic callus of dehydration and recovery wereconducted by RNA-seq (quantitation) expression profiling. Differentiallyexpressed genes between the EC of control and dehydration were3027intotal, the number of upregulated genes was1671, the number ofdownregulated genes was1536, accounted for52.10%and47.90%respectively. Differentially expressed genes between the EC of dehydrationand recovery were5737in total, the number of upregulated genes was2315, the number of down-regulated genes was3422, accounted for40.35%and59.65%respectively. Significant GO enrichment analysisshowed that the differentially expressed genes mainly enriched in:exogenous stimulus response including517DEGs, stress responseincluding302DEGs, plant hormone signal including207DEGs,transductiontranscriptional regulation, endocytosis, glucose metabolism,secondary metabolism, membrane structure, and transcription factors.Showed Agapanthus embryogenic callus related different genes expresseddurning the cryopreservation process improved cell adaptation to stress. Inthe dehydration process Agapanthus embryogenic callus response to stressperformance membrane chromatin structure, reducing substances andchanges in plant hormones, durning recovery cell stress responsemanifested in the restoration and development of related cell structureanabolic substances. Agapanthus can be optimized appropriately by addingplant hormones and reducing substances into cryopreservation system, itmay have a positive effect on improving recovery growth rate after cryopreservation. |
PDF Full Text Request