Comparing adventitious root-formation and graft-unification abilities in clones of Argania spinosa

Abstract

Argania spinosa trees have attracted attention in recent years due to their high resistance to extreme climate conditions. Initial domestication activities practiced in Morocco. Here we report on selection and vegetative propagation of A. spinosa trees grown in Israel. Trees yielding relatively high amounts of fruit were propagated by rooting of stem cuttings. High variability in rooting ability was found among the 30 clones selected. In-depth comparison of a difficult-to-root (ARS7) and easy-to-root (ARS1) clone revealed that the rooted cuttings of ARS7 have a lower survival rate than those of ARS1. In addition, histological analysis of the adventitious root primordia showed many abnormal fused primordia in ARS7. Hormone profiling revealed that while ARS1 accumulates more cytokinin, ARS7 accumulates more auxin, suggesting different auxin-to-cytokinin ratios underlying the different rooting capabilities. The hypothesized relationship between rooting and grafting abilities was addressed. Reciprocal grafting was performed with ARS1/ARS7 but no significant differences in the success of graft unification between the trees was detected. Accordingly, comparative RNA sequencing of the rooting and grafting zones showed more differentially expressed genes related to rooting than to grafting between the two trees. Clustering, KEGG and Venn analyses confirmed enrichment of genes related to auxin metabolism, transport and signaling, cytokinin metabolism and signaling, cell wall modification and cell division in both regions. In addition, the differential expression of some key genes in ARS1 vs. ARS7 rooting zones was revealed. Taken together, while both adventitious root-formation and graft-unification processes share response to wounding, cell reprogramming, cell division, cell differentiation and reconnection of the vasculature, there are similar, but also many different genes regulating the two processes. Therefore an individual genotype can have low rooting capacity but good graft-unification ability.

Keywords: adventitious roots; argan; grafting; hormone profile; transcription profile.

Figure 1

Distribution of rooting capacity among the 30 collected argan clones propagated by rooting of stem cuttings. (A) ARS1 tree. (B) Rooted cuttings from ARS1. (C) Percent rooting of all 30 clones. Black line marks 60% rooting: above this threshold, clones were considered easy to root.

Figure 2

Specific comparison between difficult- and easy-to-root clones. Two pairs of trees, ARS7, ARS2 (difficult to root) and ARS1, ARS5 (easy to root), were compared. (A) Percent rooting. (B) Percent survival. Numbers in the bars are the number of cuttings. Statistical analysis: Chi square. (C) Average number of roots per rooted cuttings. (D) Average root length. Statistical analysis (C, D): one-way ANOVA. Histological analysis of root primordia at different stages for ARS7 (E–G) and ARS1 (H). Fused roots after emergence in ARS7 (I) and ARS2 (J). Arrows in E-G show fused primordia, in H a single primordium and in I and J fused roots.

Figure 3

Hormone profiling. (A–C) Hand-cut sections and calcofluor staining showing enrichment of cambium, phloem and cortical parenchyma tissues. (A) Transverse section of the whole stem. (B) Bark after peeling, used for hormone profiling. (C) Residual inner part which was not used for profiling. C-cambium. (D–L) Hormone content analyses (ng/g wet weight) for the tissue in (B) from ARS1 and ARS7 trees. Asterisks show statistical significant difference between the samples at this time point p<0.05 One way Anova.

Figure 4

Reciprocal grafting of ARS1 and ARS7 or ARS5 and ARS2. (A) Successful grafting of ARS7/ARS1. (B, C) Results of all scion/rootstock grafting combinations. Numbers in the bars are number of repeats. Statistical analysis: Chi square, p < 0.05. (D–G) Histological analysis of the graft-unification zone in self-grafted ARS7/ARS7 and ARS1/ARS1 after 30 days (D, E) and 60 days (F, G). R-S=rootstock.

Figure 5

Bioinformatics analysis of the RNA-seq results. (A–E) RNA was prepared in three biological repeats each composed of three plants. R, rooting tissue; G, grafted tissue. The transcripts in the different groups were clustered and the content of similar or different transcripts was analyzed by Venny software in the groups showing similar cluster patterns. Red, transcripts common to rooting and grafting zones in the two trees; yellow, transcripts specific for grafting in both trees; blue and green, transcripts specific for the rooting zone in ARS7 and ARS1, respectively. (F) KEGG analysis of gene ontology according to colored zones in the Venn diagrams.

Figure 6

Expression profiles of specific genes. Expression levels were determined as number of reads in the RNA-seq using three repeats for each treatment. The best assembled hit for each transcript was translated and the amino acid sequence was aligned to that of the corresponding Arabidopsis transcript. G, grafted tissue; R, rooting tissue. (A) Expression of solitary root IAA14-like. (B) Cytokinin oxidase/dehydrogenase-like (CKX6-like), which participates in cytokinin catabolism. (C) Expansin 8-like, involved in cell wall loosening. (D) Auxin influx transporter AUX1-like. Different letters show statistically significant difference by one-way ANOVA at p < 0.05.