| Davidia involucrata Baill.,commonly known as the pigeon tree,is a deciduous tree belonging to the genus D.involucrata.It is a relic species of the ancient tropical flora from the Tertiary period and is native to China.Renowned worldwide as a precious ornamental tree,it exhibits a beautiful tree shape and distinctive white bracts surrounding its unique flowers.Propagation of D.involucrata is primarily achieved through seeds.However,its low seed setting rate,attributed to its biological characteristics,severely hinders the expansion of its wild population and its application in gardens.Thus,the exploration of effective propagation techniques has become imperative.In this study,immature zygotic embryos of D.involucrata were utilized as explants to investigate the impact of zygotic embryo developmental stage,basic medium type,and various plant growth regulators on callus induction.Through this exploration,a suitable combination and concentration of plant growth regulators were identified for inducing embryogenic callus and facilitating somatic embryogenesis.The developmental process of somatic embryos in D.involucrata was elucidated through histocytological observation.Additionally,high-throughput sequencing technology was employed to analyze the transcriptomes of embryogenic callus and non-embryogenic callus.The key findings of this study are as follows:（1）Immature zygotic embryos were collected in mid-July（approximately 11 weeks after flowering）as explants and inoculated on MS,1/2MS,and WPM basic media supplemented with 0.2 mg·L-16-BA and 1.0 mg·L-12,4-D.The highest callus induction rate of 90%was achieved on MS medium,resulting in bright light yellow,granular,and well-dispersed callus.Subsequently,the zygotic embryos were inoculated on a combination medium of 2,4-D,NAA,and 6-BA with varying concentrations and proportions.The highest callus induction rate of 94.0%was observed on MS medium supplemented with 2.0 mg·L-12,4-D and 0.5 mg·L-16-BA.The induction of embryogenic callus was most effective on the medium supplemented with 0.5 mg·L-1NAA and 1.0 mg·L-16-BA.The cells of embryogenic callus were characterized by small size,tight arrangement,clear boundaries,and large nuclei occupying almost the entire cell.In contrast,non-embryogenic callus cells were relatively larger,loosely arranged,lacking nuclei,and displayed irregular gaps between cells.（2）The embryogenic callus was cultured on a somatic embryogenesis medium to induce somatic embryogenesis.The induction rate of 22.9%was achieved using MS medium supplemented with 1.0 mg·L-1NAA and 1.0 mg·L-16-BA.Histological observations of somatic embryos at different developmental stages revealed that the development of somatic embryos followed a progression similar to that of zygotic embryos in their natural state,including the stages of globular,heart-shaped,torpedo-shaped,and cotyledon development.Mature somatic embryos successfully germinated on 1/2MS medium supplemented with 0.5mg·L-16-BA and 0.25 mg·L-1IBA,resulting in normal germination of cotyledons and radicles.Furthermore,when cultured on a medium supplemented with 1g·L-1AC,healthy plants with 2 to 3 leaflets and well-developed root systems were obtained.（3）Transcriptome sequencing of embryogenic callus and non-embryogenic callus samples generated a total of 714,945,156 reads,which were filtered and compared to obtain709,247,312 reads.Splicing the Illumina sequencing data resulted in 131,109 high-quality Unigenes.Annotation of these sequences using six major biological information databases revealed that 52,420 Unigenes（39.98%）were successfully annotated with gene functions.A total of 12,806 significantly differentially expressed genes were identified.Specifically,in EC1 treated with 1.0 mg·L-12,4-D+0.2 mg·L-16-BA compared to NEC treated with 1.5mg·L-1TDZ,8,516 genes were up-regulated and 4,569 genes were down-regulated.In EC2treated with 0.5 mg·L-1NAA+1.0 mg·L-16-BA compared to NEC treated with 1.5 mg·L-1TDZ,2,474 genes were up-regulated and 2,485 genes were down-regulated.In EC3 treated with 1.0 mg·L-1NAA+1.0 mg·L-16-BA compared to NEC treated with 1.5 mg·L-1TDZ,3,905 genes were up-regulated and 3,553 genes were down-regulated.ENrichment analysis identified 20 significantly e Nriched Gene Ontology（GO）terms,and 16,027 Unigenes（12.22%）were annotated in the KEGG database,with 11,034 participating in 19 KEGG pathways.A total of 466 transcription factors from 29 different families were predicted among the 12,806 differentially expressed genes.Several genes related to plant growth regulation,stress response and somatic embryogenesis,such as Aux,IAA,ARF,GH3,AHP,ARR,CYCD,AGL,AIL,BBM,WUS and SERK,were screened,which may be related to the regeneration ability of D.involucrata.In addition,qRT-PCR technology confirmed the expression trend of 16 differentially expressed genes in D.involucrata,and verified the transcriptome data.This study achieved the successful induction of somatic embryogenesis in D.involucrata,leading to the successful regeneration of plants.It marks the first successful induction of somatic embryogenesis in this species.Furthermore,a preliminary culture program for the regenerated plants through somatic embryogenesis was established.Additionally,the molecular mechanism underlying the formation of embryogenic callus was discussed.The outcomes of this research serve as a foundation for the conservation of germplasm resources,genetic enhancement,and the rapid propagation and dissemination of exceptional germplasm in D.involucrata.These findings have significant implications for the preservation and improvement of this species. |
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