Establishment Of The Chromosome Production System Of Neolamarckia Cadamba

Neolamarckia cadamba,an evergreen broad-leaved tree belongs to Rubiaceae family,is an important fast-growing forest with high application prospect and development value.To date,the research of Neolamarckia cadamba is mainly focused on germplasm resources,tissue culture and establishment of rapid propagation system,medicinal value development.However,the study on cytology has never been reported,and thereby the genetic background is not yet clear.In our study,we used the chromosome production method to study the chromosome number of Neolamarckia cadamba,which will lay foundation for whole genome sequencing and the construction of genetic map.Based on the established of the chromosome production system of Neolamarckia cadamba,a suitable method for chromosome observation of Neolamarckia cadamba was obtained.Furthermore,the chromosome number of Neolamarckia cadamba was determined in our study.The main steps are as follows:1.The root tips of tissue culture seedlings in Neolamarckia cadamba were used as materials for chromosomal tableting.We collected the root tips,at 9:00-10:00 in the chromosomes were divided into 4.7 % on the middle phase at that time.The length of the growth of the root tip of Neolamarckia cadamba should not be too long,with a range of 3-6mm.2.The 0.10 % colchicine solution and the 2 mM 8-hydroxyquinoline equal volume mixture were used as the pretreatment solution.The root tips were treated with the pretreatment solution at 25 ? fot 3 h had the best performance,which the chromosomes could be dispersed well and counted easily.3.75 mM KCl was used as a pre-hypotonic solution,to treat root tips.The result showed that incubation at 25 ? for 30 min would bring best effect,with chromosomedistributing in the entire cell.the best effect when processed at 25 ? for 30 min tableting,chromosome flooding in the entire cell.4.When the root tips were fixed by freshly prepared Carnot fixative at 4 ? for 24 h,the cell is best,which the cytoplasmic background is clear.5.Using 1 M hydrochloric acid as a dissociating reagent,to dissociate the root tips for6 min at a constant temperature of 60 ? in a water bath,the cell wall is softened,and the cell dispersibility is best when tableting.6.It showed no significantly difference when the root tips were post-deionized with water and 75 mM KCl for 30 min.Therefore,we used deionized water as a post-hypotonic for its high efficiency.7.Carbama magenta solution was used as the dyeing agent for staining for 2 min,which make the best performance.The nucleus was purple,and the cytoplasm was nearly colorless.8.The chromosome number of the somatic cell of the Neolamarckia cadamba is2n=44.According to the previous research on the Rubiaceae,it is assumed that the base X=11.Therefore,N.cadamba is a tetraploid species.