| Plant suspension cells can provide a simplified model system for plant research.Its development and application are mainly concentrated in herbaceous plants and crops,and the application research in forest trees is rarely reported.Compared with herbaceous plants,woody plants have obvious secondary growth characteristics,which makes them different in stress response regulation and signal transduction regulation.Therefore,in many scientific research and exploration,herbaceous suspension cell lines have obvious limitations and cannot completely replace woody plant cell lines for research.Populus simonii × P.nigra was artificially bred by breeders in the 1950s and 1960s.It is widely planted because of its fast growth,strong adaptability,cold,drought and salt tolerance.In this paper,Populu.s simonii × P.nigra was used as the research material.Through callus induction of anthers of Populus simonii × P.nigra and screening of haploid population,the haploid cell line Qu-1 with excellent traits was obtained.The application of Qu-1 in molecular biology was studied in depth,and a systematic application system of Populus simonii × P.nigra haploid cell line was established.It revealed that Populus simonii × P.nigra haploid cell line had broad application prospects and values,which could provide excellent experimental materials for plant field,especially forest molecular biology research.The main research results include:(1)The establishment of haploid callus induction system of Populus simonii × P.nigra:By observing the phenology of Populus simonii × P.nigra in this area,stamens of Populus simonii × P.nigra began to sprout in March every year,and anthers were observed at different developmental stages in mid-April.The anthers of Populus simonii × P.nigra at different developmental stages were used as explants for callus induction after routine disinfection.The obtained callus was subcultured,flow cytometry ploidy detection,k-mer analysis,and induction of complete plant regeneration.Through the statistics of callus induction efficiency,a total of 3000 callus were obtained.At the same time,it was found that the induction efficiency was the highest when the anther was in the uninucleate stage.After hybridization and ploidy detection,300 haploid calluses were finally obtained,of which 20 haploid calluses had obtained complete plants.(2)Establishment of the suspension culture system of Populus simonii × P.nigra haploid cell line Qu-1:The callus Qu-1 with light yellow color,loose texture and vigorous growth was selected from the haploid callus of renewable plants as the suspension culture material.The most suitable suspension culture conditions were screened by adjusting the proportion concentration of different hormones.The results showed that the optimal suspension culture medium of Populus simonii × P.nigra haploid cell line Qu-1 was MS+1.25 mg/L 2.4-D?0.2 mg/L KT+50 g/L sucrose,pH 6.0,the optimal culture temperature and rotation rate were 26?,110 rpm,the optimal inoculation amount was 0.02 g/mL,and the culture cycle was 10 d.(3)Study on the growth characteristics of Populus simonii × P.nigra haploid cell line Qu-1:Compared with BY-2 cell line in solid culture and suspension culture,the growth,dispersion and cell size of Qu-1 cell line were analyzed.The results showed that Qu-1 cell line showed more vigorous growth rate and good dispersion than BY-2 cell line in suspension culture.At the same time,lignin composition analysis and plant regeneration induction were carried out on Qu-1 cell line.The results showed that the total lignin content of Qu-1 cell line was 8.33%and the S/G ratio was 3.51.After adventitious bud induction,Qu-1 cell line can be regenerated to form complete plants.(4)Genome-wide sequencing of Populus simonii × P.nigra haploid cell line Qu-1:Genome sequencing of Qu-1 cell line adopts the combination of the third-generation PacBio and the second-generation sequencing.It relies on the long reading of the third-generation sequencing to ensure more complete genome assembly,and uses the second-generation sequencing data correction to ensure more accurate and reliable assembly results.At the same time,combined with new technologies such as Hi-C,the genome of Qu-1 cell line was assembled at the chromosome level,so as to provide accurate and comprehensive genomic information-for subsequent analysis.The final assembled Qu-1 cell line had a genome size of 397.9 Mb,a contig N50 of 6.7 Mb,a scaffold N50 of 20.6 Mb,and a GC content of 33.8%.The final annotated repeats were 196.327,743 bp,accounting for 49.3%of the total genome sequence.(5)Establishment of transient transformation system of Populus simonii × P.nigra haploid cell line Qu-1:In the transient transformation operation mediated by Agrobacterium,the effective transient transformation system of Qu-1 cell line was established by single factor comparison test of several main factors in transient transformation of Qu-1 cell line.The results showed that the transient transformation effect of-Qu-1 cell line was the best when the Qu-1 cell line cultured for 6 days was co-cultured with Agrobacterium solution containing 0.2 OD600 of water as solvent,0.75 mM AS and 0.0002 mg/L TDZ infection suspension at 26?and 110 rpm for 2 d;In the transient transformation mediated by protoplasts,Qu-1 cell line cultured for 8-9 d was selected for protoplast isolation.The protoplast yield was the highest.and the transformation efficiency was up to 30-35%.A variety of organelles localization and BiFC tests were carried out using the Qu-1 protoplast isolation and transformation system.(6)Establishment of the stable transformation system of Populus simonii × P.nigra haploid cell line Qu-1:The over-expression of Qu-1 cell line,RNAi inhibition expression and CRISPR/Cas9 gene editing genetic transformation test were carried out,and the conventional molecular detection of resistant callus was carried out.The results showed that resistant callus could be obtained after 25-30 d of resistance screening.DNA molecular level detection showed that exogenous genes had been integrated into the genome of Qu-1 cell line,and RNA molecular level detection showed that the relative expression levels of over-expression and RNAi-suppressed genes in different transgenic callus were different.Transgenic callus obtained by gene editing were homozygous mutants.(7)Analysis of response of Populus simonii × P.nigra haploid cell line Qu-1 to ABA treatment:Different concentrations of ABA were set to treat Qu-1 cell line for different time.The activity of Qu-1 cell line after treatment was detected,and the reported response genes were quantitatively analyzed to determine the optimal concentration of ABA treatment,so as to establish the ABA treatment system of Qu-1 cell line.Qu-1 cell line was treated with 100 ?M ABA for 0 h,0.5 h,4 h,12 h and 24 h,and transcriptome analysis was performed.The results showed that Qu-1 cell line had the same response pathway as known ABA.In view of the simple and convenient operation of stress treatment,it can be used as an excellent model system for stress treatment.In summary,the development and application of haploid cell line Qu-1 in Populus simonii× P.nigra showed that it had great potential to become a model cell line for plant research,especially for forest molecular biology,and provided important help for related fields. |
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