Study On The Biological Characteristics Of Recalcitrant Seeds And Mechanisms Of Desiccation Sensitivity Of Taxillus Chinensis(DC.)Danser

Taxillus chinensis(DC.)Danser is a well known traditional Chinese medicine which is difficult to breed.The seeds of Taxillus chinensis are typical recalcitrant seeds with fast inactivation and high sensitivity dehydration.What's more,as the only reproductive materials,these characteristics of seeds seriously restrict its breeding.At present,there are many studies on the preservation of recalcitrant seeds,however,little successful case is presented.The main reason of this issue is that the mechanism of death caused by dehydration is not clear.Thus,it becomes an important scientific problem that seeds desiccation sensitivity leads to recalcitrant seed death.The aim of this research project is to realize the short and long term aim of preservation of Taxillus chinensis seeds by studying its biological characteristics,dehydration sensitivity,cryopreservation etc.The morphological structure and anti-oxidation system of the cells during the dehydration process of Taxillus chinensis seeds were thoroughly studied.Screening the seeds of dehydration sensitive critical period for high-throughput Solexa sequencing.The key genes related to the sensitivity of dehydration were excavated and verified by RT-PCR,which revealed the molecular mechanism of dehydration sensitivity of Taxillus chinensis seeds.The study not only provides theory basis for the key technology in the preservation of Taxillus chinensis seeds,but also provides new knowledge and ideas for other recalcitrant seed's preservation.The main findings are as follows:(1)The factors affecting the seed viability of the parasite are the dyeing time>TTC concentration>dyeing temperature.The best method of identification is cut longitudinally along the two sides of the long axis of the seed.Then the seeds are placed in a brown jar with 0.8%TTC solution and stained in a 30? incubator for 8h.(2)The seeds of the most suitable germination bed are paper beds.The difference of seed germination rate between different temperature treatments is significant,and the optimum germination temperature was variable temperature group(25/30?).Light has no effect on seed germination.Seeds without disinfection,sterilization treatment with the best results.The quality of the seeds of different batches is different,and the spring seed is better than autumn and winter seed.(3)Taxillus chinensis seed is a typical recalcitrant seed,which is extremely sensitive to low temperature.It is difficult to long-term preservtion,as the seeds lose their vitality after 7 days natural state preservation.The best preservation method is that the fresh seeds treated with sterile water were soaked in 2 mg/L ABA solution for 30min.The drained surface water was removed,while the seeds are spread out in a Petri dish with 2 layers of sterile filter paper and packed in a sealed bag placed in the refrigerator at 4?.(4)According to the gerrmination characteristics of Taxillus chinensis seeds,the quality of sage seeds is divided into three grades.For first grade,purity was 100%,seed germination rate>75.0%,water content>50%,hundred-grain weight>60.0 g;for second grade,purity was 100%,seed germination rate>70.0%,water content>40%,hundred-grain weight>50.0 g;for third grade,purity was 100%,seed germination rate>60.0%,water content<40%,hundred-grain weight<50.0 g.(5)The best breeding method:fresh and full fruit without plant diseases or insect pests is collected.After the peel is removed,the seed is stick in mulberry branches greater than 1.0 cm in diameter.Mulberry forest air humidity is above 80%.(6)It is clear that the viability and germination rate of Taxillus chinensis seeds were associated with the water content in seeds.The germination rate of the fresh seeds was 86%(viability:99%),which was dropped to 66%(viability:66%,moisture content:35.17%)after being dehydrated for 16 hours.If the seeds were controlled with dehydration for 36 hours,the moisture content was decreased to 24.93%,the viability was decreased to 15%and the germination rate was 6%.After 40 hours' dehydration the moisture content,viability and germination rate were examined as 23.47%and 9%respectively with no germination.(7)The changes of cell microstructure and ultrastructure of Taxillus chinensis seed dehydration process greatly.Microscopic observation revealed that the dehydration process of the cell gap increased gradually,the gradual separation of cytoplasm and cell wall,which leads to further degradation of cell.Ultrastructure showed various organelles in the dehydration process affected by different degree of damage,mitochondrial structure rupture,nuclear envelope became blurred,nucleolus gradually degraded starch and fat body decomposed further deterioration.The change of physiological index during dehydration become larger.With the increase of dehydration time,the contents of ABA,ZR,GA and IAA of endogenous hormones showed a trend of increasing first and then decreasing.Seed SOD activity,CAT activity and ASA-POD activity showed an overall downward trend.At the same time,the content of superoxide anion and the content of MDA increased.(8)The different extraction methods of total RNA of Taxillus chinensis in different parts have been studied.Results shows TriZol kit and modified TriZol method can not effectively remove mistletoe polysaccharides and proteins and other impurities,CTAB-LiCl method was considered to be the best favorable methods for RNA isolation from different sections.(9)Initially,Taxillus chinensis seeds were characterized as recalcitrant seeds.Then,biological replicates of fresh loranthus seeds(CK),and seeds after being dehydrated for 16 hours(Tac-16)and 36 hours(Tac-36)were sequenced by RNA-Seq,generating 386,542,846 high quality reads.A total of 164,546 transcripts corresponding to 114,971 genes were assembled by Trinity and annotated by mapping them to NCBI non-redundant(NR),UniProt,GO,KEGG pathway and COG databases.Transcriptome profiling identified 60,695,56,027 and 66,389 transcripts(>1 FPKM)in CK,Tac-16 and Tac-36,respectively.Compared to CK,we obtained 2,102 up-regulated and 1,344 down-regulated transcripts in Tac-16 and 1,649 up-regulated and 2,135 down-regulated transcripts in Tac-36 by using edgeR.Among them some have been reported to function in dehydration process,such as RD22,heat shock proteins(HSP)and various TFs(MYB,WRKY and ethylene-responsive transcription factors).Interestingly,transcripts encoding ribosomal proteins peaked in Tac-16.It is indicated that HSPs and ribosomal protei may function in early response to drought stress.Raw sequencing data can be accessed in NCBI SRA platform under the accession number SRA309567.In this study,the biological characteristics of Taxillus chinensis seeds were systematically studied.This is the first time to profile transcriptome globally in Taxillus chinensis seeds.Our findings provide insights into the gene regulations of Taxillus chinensis seeds in response to desiccation and expand our current understanding of drought tolerance and germination of recalcitrant seeds.