| Pinus massoniana Lamb.is an important afforestation tree species with a wide distribution in the subtropical region of southern China,used for both resin and timber.The induction of organogenic plant regeneration from adventitious buds of masson pine is one of the basic means of preserving and expanding the use of excellent forest tree germplasm.However,how to induce more high-quality adventitious buds in a short period of time is still one of the technical bottlenecks in the construction of masson pine tissue culture system.The concentration of plant growth regulators(PGR)and the level of induction duration have an important influence on the induction of adventitious buds.In this study,we used plant tissue culture and transcriptome sequencing techniques to investigate the differential morphological effects of different concentrations of 6-BA and different induction period on the adventitious buds induction of masson pine,and transcriptome sequencing analysis was carried out for the four groups of adventitious shoots with differential phenotypes that treated with different 6-BA concentrations and induction period.To explore the expression patterns of genes related to the induction of adventitious shoots in masson pine under different 6-BA concentrations and different induction durations.The main findings are as follows.1.A set of significantly cross-level interaction effects of 6-BA concentrations and induction duration on adventitious bud induction and elongation have been observed.The number of adventitious buds per explant increased with the increase of 6-BA concentrations at specific duration(2d to 3 2d),and the adventitious buds multiplication coefficient was increased from 0 to 7.78.Meanwhile,the number of adventitious buds per explant increased firstly and then decreased with the increase of induced duration from 2d to 32d at a specific 6-BA concentration(4.4 μmol·L-1,11.1μmol·L-1,44.4μmol·L-1),and a turning point was observed at 16d with the largest number of adventitious buds grew normally.In addition,the adventitious buds was shortened with the increase of 6-BA concentration or the prolongation of induction duration.2.The results of the axillary bud proliferation and elongation culture of the stem segment of masson pine show that the induction and proliferation of axillary buds on stem segments were significantly influenced by the concentration of 6-BA and the inducted duration.The number of axillary buds proliferated with increasing 6-BA concentration and induction time,and after 32d of induction at the concentration of 22.2 μmol·L-1.Its axillary bud multiplication coefficient is 6.11,and the maximum number of axillary buds proliferated in a single stem segment was 36～40.The ratio of ammonium to nitrate in WPM medium is 0.2～0.4 and the treatment of removing NH4+can promote the elongation of axillary buds to a certain extent.The maximum growth of axillary buds after 30 days of culture is 0.62cm and 0.53cm,respectively,which were higher than the treatment group.The addition of a suitable IBA concentration of 0.98 μmol·L-1 has a better effect on promoting the elongation and growth of axillary buds,and the growth of axillary buds per 30 days is 0.42cm,while other concentration treatments have no obvious effect on promoting their elongation and growth.3.Different concentrations of IBA have a significant effect on the induction of adventitious roots of stem buds.As the IBA concentration increases(4.9 μmol·L-1,9.8 μmol·L-1,19.6 μmol·L-1),the rooting rate first increases and then decreases(31.82%,48%,45.45%),and the adventitious root state also has different changes.The adventitious buds rooted well under 9.8 μmol·L-1 IBA,with a large number of fine roots observed,then the regenerated seedlings survived when they were transferred in pots.4.Transcriptome analysis of the mature embryo explants under these 4 groups of low concentration at 6-BA 4.4 μmol·L-1 or high concentration at 6-BA 44.4μmol·L-1,both them with induction period of 4d or 32d.The results showed that the 12 sequencing samples were assembled and assembled to obtain a total of 293,900 unigenes,and 110,781 unigenes were obtained after clustering and de-redundancy.With a threshold of FDR<0.01 and |logFC|>2,there were 3425 genes differentially expressed during the different induction treatments.Differentially expressed genes were found to be significantly enriched by GO and KEGG analysis with thresholds of Qvalue<0.01 and Qvalue<0.05,respectively.When the inoculated explants respond to environmental stress caused by the concentration and duration of PGR,there are significant differences in the biological processes such as stimulus response,stress response and defence response.The concentration and duration of PGR are closely related to the differential expression of related genes in pathways such as plant hormone signal transduction,phenylpropane biosynthesis and biosynthesis of secondary metabolite biosynthesis.In the plant hormone signal transduction,AHK,AHP,ARR-As and other cytokinin-related genes were up-regulated under the induction treatment of adventitious shoot short duration(4d),and down-regulated under the induction treatment of adventitious shoot long duration(32d).However,the expression of auxin-related genes such as AUX/IAAs,AUX/LAXs,SAURs,GH3s and ARFs during the adventitious bud induction process is reversed,and the expression is down-regulated under short-term induction treatment(4d),and up-regulated under long-term induction treatment(32d). |
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