This study were conducted to identify the exact time on which MT promotes AR formation arising from tissue culture plantlets of Malus prunifolia apple rootstocks, in the study, we set five treatment groups: MT, MT0-2, MT2-5, MT5-20 along with one control group (Fig. 1A). From Fig. 1B, No morphological changes were evident in all groups within 5d, however on 10d ARs emerged from stem basal parts. At 20d, the number of AR was highest in the group of MT0-2 than other groups, in group of MT2-5 and MT5-20 were more conducive to AR formation than control and MT group at 20 d, but there is no difference between them. In order to observe anatomical at the different stage of AR formation, sectioning paraffin-embedded samples was made and stem sections was viewed by the light microscope. On 0 day cross sections of the samples exposed the existence of competent cells, but the capability of cambial cells to undergo mitosis was observed at 5d, now cell divisions were appeared in the agminated cells, AR appeared on transection of stem basal at cultured in medium 10 days (Fig. 1C).
Furthermore, we also measured the root rate, number of AR, crossings, root length, root surface area and root volume between five groups, it was consistent with the phenotype observations of AR formation, The results showed that all measured parameters were higher in MT0-2 group as compared to other groups, the control group was the minimum number of ARs, and other root measured parameters as compared with other groups (Fig. 2). The result showed that MT mainly promoted the AR formation at 0-2d during AR induction stage.
ARs were classified into 3 groups based on their diameter: 0–2.0 mm, 2.0–5.0 mm and > 5.0 mm. According to the data of AR number, length and surface area, the categories of 0–2.0 mm constituted most percentage of total root, differ from these indexes was root volume, the categories of 2.0–5.0 mm was the largest than other classes in all groups (Fig. 3). The data were showed that the MT0-2 group was the greatest than other groups, there were twice as many in the MT0-2 group as in the control group in the 0–2.0 mm class (Fig. 3). We can draw conclusion that the most of AR was fine root (0–2.0 mm).
The contents of hormones: MT, IAA, ZR, GA1 + 3 and ABA were analyzed in MP tissue culture plantlets after being treated with MT, as can be seen in Fig. 4, MT content was visibly greater in MT0-2 group than other treated groups during early developmental stage of ARs, and reached a peak at 5 d in MT0-2 group. In group of MT and MT0-2, IAA, ZR and GA1 + 3 content were apparently higher than control group during AR induction at 1d and 2d, but their content were apparently lower in MT0-2 than other group at 10d, ABA content was approximately contrary to content of IAA, ZR and GA1 + 3 (Fig. 4).
Expression of MT and IAA signal transduction associated gene in response to MT were analyzed, expression of MdTDC1, MdHIOMT2, MdASMT1 and MdASMT2 appeared the similar regulation of expression, their expression were obviously greater in MT0-2 than other groups expect at 0 and 20d, MdSNAT and MdHIOMT1 expression were higher in MT0-2 than other groups at 1d, 5d and 20d (Fig. 5).
In further, to determine whether there is a link between MT and IAA, we measured the IAA related gene expression, related gene of IAA synthesis and signal transduction MdYUCCA2, MdYUCCA10, MdARF7 and MdARF19 were visibly greater in MT0-2 than other groups at 1d, 2d and 3d, IAA transport related gene MdAUX1, MdPIN1, and MdPIN3 were higher in MT0-2 than other groups at 2d, however, IAA signal transduction related gene MdIAA5 was lower in MT0-2 than other groups during AR induction stage (Fig. 6).
To investigate whether MT could affect cell division,expression of genes involved in cell cycle MdCYCD1;1 and MdCYCD3;1 were analysed, which were highly expressed in MT0-2 at 3 d and 10d. Now it can be concluded that application of MT promoted AR formation in apple rootstock, qRT-PCR analysis showed that all of root development related genes were higher at most time point after MT treatment. We found among all root related gene MdWOX11 expression in MT treated group was 5.6 times higher than control group at 2d (Fig. 7). This suggests that in the stage of AR induction, MdWOX11 are likely to have crucial roles in AR induction after treated with MT.
The expression of MdWOX11 was induced by IBA treatment (Fig. 7). We received the over-expressed (OE) transgenic lines of MdWOX11-OE15#, 16#, 20# in ‘GL3’, identification of DNA level of overexpression MdWOX11 transgenic lines were list in Figure S1. In order to confirm whether MdWOX11 transgenic lines exhibited better response to MT signalling, wild-type and transgenic apple tissue culture plantlets were treated with 0.7 mg.L− 1 IBA served as a control, another group was treated with MT at 0-2d which named as MT0-2. The number of AR were higher in the group of MT0-2 than control group, both of the over-expressed MdWOX11 transgenic lines and ‘GL3’, over-expressed MdWOX11 transgenic lines showed more ARs than those of ‘GL3’ (Fig. 8A), the rate and number of AR were conformed to the phenotype of these apple plants (Fig. 8B and C). MdWOX11 over-expressed plants were more sensitive to exogenous MT treatment than wild type (Fig. 8), these result indicated that MdWOX11 induced AR formation in response to MT treatment.