Establishment Of Xylem Cells Capture System By Laser Microdissection In Neolamarckia Cadamba

Neolamarckia cadamba,a fast-growing broad-leaved tree species of the genus Rubiaceae with dense texture,is widely distributed in the tropical and subtropical regions.The rapid growth of N.cadamba depends on the development process of the primary and secondary xylem cells.Therefore,understanding the molecular regulatory mechanism of the xylem growth and development will be of great importance in the genetic improvement and application of tropical and subtropical fast-growing species.It is an effective way to explore the key genes responsible for xylem growth and development by comparing the expression profiles of xylem cells at different stages.However,it has difficulty in obtaining high-quality specific tissues or cells in woody plants.Laser microdissection(LMD)allows for the location and isolation of specific tissues or cells like xylem from heterogeneous tissues under a microscope with the assistance of a laser beam.In this study,the parameters of LMD were optimized and high-quality RNA from xylem cells of N.cadamba at different stages was isolated.The main results are as follows:1.The seedlings of N.cadamba from tissue culture and greenhouse were used as candidate materials for cryo-section analysis.The result showed that clear cell slides were obtained and high-quality RNA was isolated when using four-month-old greenhouse seedlings with semi-embedded and liquid nitrogen frozen treatments.Therefore,four-month-old seedlings growing in greenhouse were used for the further study.2.The paraffin sections analysis of different stem sections of the greenhouse seedlings revealed that the second stem section is a transition node from primary growth to secondary growth.Hence,we took the first,second and fourth stem sections as materials for dissection of xylem cells.3.The parameters of laser microdissection for the xylem cells were optimized,including power 37?Aperture 1?Speed 7?Head Current(emission intensity)100%?Pulse Frequency1101.The total RNA from xylem cells of the first,second and fourth stem segments was extracted using the Rneasy Micro kit(Qiagen)kit.RNA integrity was evaluated using a Bioanalyzer 2100,which rRNA Ratio [25S/18S] was higher than 1.2 and RNA Integrity Number [RIN] was higher than 6.1.The results suggested that RNA from the xylem cells could be used for high-throughput sequencing.