Somatic embryogenesis process of birch
To observe the somatic embryogenesis of birch trees, we used microscopic techniques. First, sterile birch tissue culture seedlings were cultured (Fig. 1a). Cut 1-2 cm stem fragments of birch into callus induction medium(CIM)(Fig. 1b). SE was induced in differentiation medium after callus formation (Fig. 1c). Through the induction of somatic embryos in birch, we observed the development of somatic embryos. globular embryo (Fig. 1d, h), heart-shaped embryo (Fig. 1e, i), torpedo embryo (Fig. 1f, j) and cotyledon embryo (Fig. 1g, k). The microstructure showed that the developmental structure of somatic embryos was relatively close, and it was difficult to separate a single embryo.
Overexpression of AtWUS resulted in abnormal somatic embryogenesis
To understand the role of the AtWUS gene in stem cell regulation and growth control in birch. We constructed AtWUS overexpression vector. The expression was under the cauliflower mosaic virus coat protein promoter, resulting in the universal expression of adjacent genes. We observed the difficulty of somatic embryogenesis and bud formation in nontransgenic calli (Fig. 2a, c, g). However, the explants overexpressing the AtWUS gene showed more somatic embryogenesis (Fig. 2d, e, f). Clustered buds were also formed on the callus (Fig. 2h, i, g). This phenomenon indicated that overexpression of heterologous AtWUS promoted the development of somatic embryos and formed more buds.
It has been reported that the AtWUS gene can promote SE, but the efficiency of promoting somatic embryogenesis is different. According to the somatic embryogenesis rate of explants. We observed that the highest transgenic line 3 (L3) reached 717.1%, which was approximately 7 times higher than the 101.4% of wild-type plants (Table 1). The somatic embryogenesis rates of L1 and L2 were 615.2% and 371.6%, respectively (Table 1), which were higher than those of the wild type. This indicates that overexpression of AtWUS can increase the incidence of somatic embryos in birch.
Transformation from calli to somatic embryos depends on AtWUS overexpression
To characterize the effect of AtWUS gene overexpression on somatic embryogenesis in birch. We analyzed the expression of several candidate genes in transgenic and wild-type AtWUS L3 plants. We isolated callus and early globular embryo stages of somatic embryogenesis and analyzed gene expression (Fig. 3a-d). qPCR was used to determine the expression level of AtWUS in birch. The calli of transgenic plants (L1, L2, L3) cultured in SIM were analyzed, and wild-type (WT) birch calli were used as controls. The data showed that AtWUS was overexpressed in different transgenic lines (Fig. S1a).
Expression of the AtWUS gene induces the lateral wall of roots to form bud meristems(Gallois, et al. 2004; Negin, et al. 2017). The expression of the BpWUS gene in calli and globular embryos was lower than that in control plants, which may be due to the high homology and similar structure of BpWUS and AtWUS. The expression of the endogenous BpWUS gene was inhibited (the amino acid sequence alignment of the gene in this paper is shown in Fig. S3). STM acts upstream of WUS, WUS and STM to complement each other to activate cell division and control the formation of bud meristem (Endrizzi, et al. 1996; Gallois, et al. 2002; Lenhard, et al. 2002). The BpSTM gene was highly expressed in the formation of globular embryos, but there was no significant difference between the wild type and the callus. This indicated that the overexpression of the atwus gene and BpSTM gene interacted to control the formation of the meristem and promote the generation of somatic embryos.
LEC1, LEC2, ABI3 and FUS3 are essential for SE (Horstman, et al. 2017). The expression of LEC1 and FUS3 can induce microspore embryogenesis (Ikeda-Iwai, et al. 2002). Our results showed that the expression of AtWUS activated the high expression of BpLEC1 and BpLEC2 in calli and globular embryos (Fig. 3e). The high expression of BpABI3 and BpFUS3 was further activated (Fig. 3e). This explains the abnormal increase in somatic embryos. CUC2 is the gene required for STM expression, but there was no significant difference in BpSTM expression between calli and controls, and c BpCUC2 was highly expressed. The opposite is true in globular embryos. This may be due to the disorder of endogenous gene regulation caused by overexpression of the AtWUS gene. Auxin plays an important regulatory role in plant embryonic development. We studied auxin transport BpPIN1. The BpPIN1 gene was highly expressed in globular embryos but not in calli. The expression of the PIN1 gene was activated by an auxin concentration gradient after somatic embryogenesis(Su, et al. 2009) (Fig. S1b).
We counted the quality of transgenic birch and wild-type control for 40 days. Abnormal proliferation of somatic embryos was found to have significant quality differences. These quality differences represent the abnormal proliferation of somatic embryos (Table S1).
The overexpression of AtWUS led to an abnormal increase in the number of lateral branches and bud meristem of birch
Through the cultivation of transgenic plants, we found that transgenic plants had developed lateral branches (Fig. 4a). The average lateral branch was 5.38 ± 0.49. Through 150 days of culture, we monitored plant height every 10 days for statistical analysis (Fig. 4a-c, e). The plant height difference between the initial wild-type and transgenic plants was small. As plants grew, wild-type plants grew faster than transgenic plants. This may be caused by nutritional limitations. More lateral branches require more nutrition, which limits the development of transgenic plants. We cultured the plants for 150 days to determine the shoot meristem number of the 2 cm part of the stem. The statistical results showed that the wild type was 1.29 ± 0.69. However, the transgene was 5.28 ± 1.25. This indicated that overexpression of AtWUS resulted in an abnormal increase in bud meristem (Fig. 4f-h). Buds change from a single form to an axisymmetric form (Fig. 4f, g).
To further analyze the causes of abnormal increases in lateral branch number and bud meristem of birch. We identified transgenic birch. The PCR results of the AtWUS gene in transgenic plants showed that 882 bp had a specific amplification fragment of the AtWUS gene (Fig. 5a). We used 35S:WUS gene fusion PCR detection to show a 1200 bp fusion amplification fragment (Fig. 5b). Fig. 5c shows the southern blot results of AtWUS L3. We used digoxin-labeled AtWUS gene probes (see Fig. S5 for electrophoresis). The identification of GFP in birch roots showed that AtWUS was successfully expressed (Fig. 5d).
AtWUS regulated gene expression resulting in abnormal shoot meristem increase
To further explain the abnormal increase in bud meristem on birch transgenic paper. We observed the shoot meristem characteristics of wild-type and transgenic plants using frozen sections (Fig. 6a-d). The bud meristems were all single, and there were no abnormal differences (Fig. S4). The expression of genes related to the BpWUS gene in the bud meristem was analyzed. The results showed that the expression of the BpWUS gene was downregulated. The expression levels of BpLEC1, BpLEC2 and BpFUS3 were upregulated, and the expression of BpABI3 was not significantly different (Fig. 6e). The expression levels of BpSTM and BpCUC2 were upregulated. The auxin transport-related gene BpPIN1 was significantly upregulated. The results showed that the overexpression of AtWUS activated BpWUS-related regulatory genes and induced the formation of meristems on shoots (Fig. S2).