Establishment Of The Technical System That Precisely Isolates Citrus Nucellar Embryo Initial Cells Using Laser Microdissection

Polyembryony, which apomictic embryos develop in addition to the zygotic embryo in a seed, is common in many citrus cultivars. Nucellar embryo which is unique and stable is the genetic breeding research forcus of citrus. Nucellar embryos initiate from the nucellar embryo initial(NEI) cells surrounding the developing sexual embryo sac. Whereas, most studies on nucellar embryo take the whole ovule as the research object without considering the effect of different cell types in one ovule, leading to the limited knowledge on the transcriptional regulation of citrus nucellar embryo initiation. Laser Microdissection(LMD) is an advanced technology used to isolate desired and less accessible cells for expression profiling of genes and proteins from tissue sections with the assistance of the laser. It is successfully used in animal and plant research field, because of its specificity and high efficiency. With the development of this technology, capturing of NEI cells separately was practicable. Therefore, isolating of high quality RNA from NEI is the key step for underlying the transcriptional regulation of citrus nucellar embryo initiation. The results are as follows:1. Through Citrus sinensis ?Valencia‘ fruit paraffin sections, we observed nucellar embryo initiation and development process. 7 DAF, we first observed NEI cells histologically, cells with thickened walls and dense protoplasm surrounded the embryo sac near chalazal. From serial sections,21 DAF, the initial cells become larger and locate in distinct clusters in ovule. Until 35 DAF, NEI start dividing and subsequently relocating inside embryo sac. Nucellar embryos experience a fast development stage. 63 DAF, a few globular embryos can be observed, embryogenesis become asynchronous. In later stages, Hearted-shaped embryo and Cotyledon-shaped embryo can be observed.2. Comparison the effect of fruit paraffin sections of five different polyembryony varieties at the stage of 10 DAF, on unstained sections, we found that in ovules of Citrus reticulata ?Ponkan‘ NEI cells aggregated around the embryo sac and were easily distinguished from the surrounding somatic nucellar cells. So we took Citrus reticulata ?Ponkan‘ as the most suitable material for dissection of NEI cells.3. Collected fruit of Citrus reticulata ?Ponkan‘ 10 DAF was first fixed in Canoy‘s fixative for 12 h, then dehydrated using graded ethanol and stored at 4℃. Samples were embedded with paraplast Plus chips(Leica Microsystems), then sectioned 8μm. LMD samples with Leica AS Laser Microdissection System before staining. Optimized technical specifications of LMD under 40×: Aperture 7, power 13,Speed 8,Head Current 98%,Pulse Frequency 1300. Altogether 10 mm2 cells were collected for RNA extraction, which was conducted with Arcturus Pico Pure RNA isolation kit(Life Technologies). RNA integrity was tested using Agilent 2100 Bioanalyzer, with an r RNA Ratio [28S/18S] of 0.5 and an RNA Integrity Number [RIN] of 4.5.