Enhancing wheat regeneration and genetic transformation through overexpression of TaLAX1

Abstract

In the realm of genetically transformed crops, the process of plant regeneration holds utmost significance. However, the low regeneration efficiency of several wheat varieties currently restricts the use of genetic transformation for gene functional analysis and improved crop production. This research explores overexpression of TaLAX PANICLE1 (TaLAX1), which markedly enhances regeneration efficiency, thereby boosting genetic transformation and genome editing in wheat. Particularly noteworthy is the substantial increase in regeneration efficiency of common wheat varieties previously regarded as recalcitrant to genetic transformation. Our study shows that increased expression of TaGROWTH-REGULATING FACTOR (TaGRF) genes, alongside that of their co-factor, TaGRF-INTERACTING FACTOR 1 (TaGIF1), enhances cytokinin accumulation and auxin response, which may play pivotal roles in the improved regeneration and transformation of TaLAX1-overexpressing wheat plants. Overexpression of TaLAX1 homologs also significantly increases the regeneration efficiency of maize and soybean, suggesting that both monocot and dicot crops can benefit from this enhancement. Our findings shed light on a gene that enhances wheat genetic transformation and elucidate molecular mechanisms that potentially underlie wheat regeneration.

Keywords: TaGRF4–TaGIF1; TaLAX1; auxin; cytokinin; genetic transformation; plant regeneration; wheat.

Figure 1

TaLAX1 promotes shoot regeneration in the common wheat variety Fielder. (A) Schematic representation of the PC186-TaLAX1-myc vector with the ZmUbi promoter and Nos terminator; the PC186 empty vector was used as a control. (B) Shoot regeneration phenotypes of immature embryos infected with the empty vector (control) or PC186-TaLAX1-A/B/D-myc vector. CIM, callus induction medium (including WLS-AS, WLS-Res, WLS-P5, and WLS-P10 media in the PureWheat transformation); SIM, shoot induction medium (LSZ-P5 in the PureWheat transformation); 0d and 42d, immature embryos were placed on CIM for 0 or 42 days; 5d and 20d, immature embryos were placed on CIM for 42 days and then transferred to SIM for 5 days or 20 days. Scale bar, 1 cm. (C) Regeneration frequencies of immature embryos infected with control or PC186-TaLAX1-A/B/D-myc vector. Regeneration frequency = no. of calli showing at least one regenerating shoot/no. of inoculated embryos × 100%. (D) Regenerating shoot frequencies of immature embryos infected with control or PC186-TaLAX1-A/B/D-myc vector. Regenerating shoot frequency = no. of regenerating shoots/no. of inoculated embryos × 100%. (E) Callus proliferation frequencies of immature embryos infected with control or PC186-TaLAX1-A/B/D-myc vector. Callus proliferation frequency = increased weight of callus after induction on CIM for 42 days/weight of immature embryos before induction. Values in (C–E) are means ± SEM from at least three independent experiments. Black points are the results from individual experiments. One-way ANOVA and Tukey’s multiple comparison tests were performed. Different lowercase letters indicate statistically significant differences (P < 0.05).

Figure 2

Overexpression of TaLAX1-A promotes shoot regeneration in different wheat genotypes. (A) Shoot regeneration phenotypes of immature wheat embryos of different genotypes infected with empty vector (control) or TaLAX1-A-OE vector. Scale bar, 1 cm. (B) Callus proliferation frequencies of wheat immature embryos of different genotypes infected with control or TaLAX1-A-OE vector. (C) Regeneration frequencies of wheat immature embryos of different genotypes infected with control or TaLAX1-A-OE vector. (D) Regenerating shoot frequencies of wheat immature embryos of different genotypes infected with control or TaLAX1-A-OE vector. CS, Chinese Spring; KN199, Kenong 199; SN28, Shannong 28; AK58, Aikang 58; JM22, Jimai 22. Values in (B–D) are means ± SEM from at least three independent experiments. Black points are the results from individual experiments. ∗∗∗∗P < 0.0001; ∗∗∗P < 0.001; ∗∗P < 0.01; ∗P < 0.05 (Student’s t-test, two-tailed).

Figure 3

The function of TaLAX1-A in regeneration is heritable. (A) Shoot regeneration phenotypes in Fielder T1 progeny of non-transgenic lines (control) or the TaLAX1-A-OE T1-8# transgenic line infected with the Ubi_pro:GUS vector. Scale bar, 1 cm. (B) Regeneration frequencies of control and TaLAX1-A-OE T1-5#, TaLAX1-A-OE T1-8#, and TaLAX1-A-OE T1-10# lines in Fielder. (C) Regenerating shoot frequencies of control and TaLAX1-A-OE T1-5#, TaLAX1-A-OE T1-8#, and TaLAX1-A-OE T1-10# lines in Fielder. (D) Transformation frequencies of control and TaLAX1-A-OE T1-5#, TaLAX1-A-OE T1-8#, and TaLAX1-A-OE T1-10# lines in Fielder. Transformation frequency = no. of transgenic shoots with GUS signals/no. of inoculated embryos × 100%. (E) Shoot regeneration phenotypes in Chinese Spring T1 progeny of non-transgenic lines (control) or the TaLAX1-A-OE T1-7# transgenic line infected with the Ubi_pro:GUS vector. Scale bar, 1 cm. (F) Regeneration frequencies of control and TaLAX1-A-OE T1-3#, TaLAX1-A-OE T1-7#, and TaLAX1-A-OE T1-11# lines in Chinese Spring. (G) Regenerating shoot frequencies of control and TaLAX1-A-OE T1-3#, TaLAX1-A-OE T1-7#, and TaLAX1-A-OE T1-11# lines in Chinese Spring. (H) Transformation frequencies of control and TaLAX1-A-OE T1-3#, TaLAX1-A-OE T1-7#, and TaLAX1-A-OE T1-11# lines in Chinese Spring. (I) Generation of TaLAX1-A knockdown lines by CRISPR–Cas9. Two target sites in the promoter of TaLAX1-A are shown. PAM, protospacer-adjacent motif. (J) Relative expression of TaLAX1-A/B/D in the wild type and two talax1-a-cr transgenic lines of Fielder. (K) Regeneration frequencies of the wild type and talax1-a-cr 1# transgenic line in Fielder. (L) Transformation frequencies of the wild type and talax1-a-cr 1# transgenic line in Fielder. Values in (B)–(D), (F)–(H), (K), and (L) are means ± SEM; values in (J) are means ± SD. All experiments were performed at least two times. Black points are the results from individual experiments. ∗∗∗P < 0.001; ∗∗P < 0.01; ∗P < 0.05; ns, not significant (Student’s t-test, two-tailed).

Figure 4

TaLAX1-A overexpression improves transformation and gene editing frequencies in the common wheat variety Fielder. (A) Shoot regeneration and transformation phenotypes of immature embryos infected with the Ubi_pro:GUS (GUS) or TaLAX1-A-OE-GUS vector. Scale bar, 2 mm. (B) Regeneration and transformation frequencies of immature embryos infected with the GUS or TaLAX1-A-OE-GUS vector. (C) Regions of the Q gene targeted with guide RNAs. PAM, protospacer-adjacent motif. (D) Shoot regeneration phenotypes of immature embryos infected with the Q-CRISPR or TaLAX1-A-OE-Q-CRISPR vector. Scale bar, 1 cm. (E) Regeneration frequencies, editing frequencies, and frequencies of gene-edited T0 shoots of immature embryos infected with the Q-CRISPR or TaLAX1-A-OE-Q-CRISPR vector. Editing frequency = no. of T0 plants with Q editing/no. of inoculated embryos × 100%. Frequency of gene-edited T0 shoots = no. of Q-gene-edited T0 plants/total no. of T0 plants × 100%. (F) Twelve transgenic T0 plants with Q-edited genomic sequences. The 2#, 8#, 9#, 12#, 16#, and 22# lines carry two target mutations. (G) Edited T0 plants showing increased numbers of florets per spikelet. Scale bar, 0.5 cm. Values in (B) and (E) are means ± SEM from three independent experiments. Black points are the results from individual experiments. ∗∗∗P < 0.001; ∗∗P < 0.01; ∗P < 0.05; ns, not significant (Student’s t-test, two-tailed).

Figure 5

RNA-seq analysis of TaLAX1-A-OE transgenic calli and activation of TaGIF1-A and TaARF3-D by TaLAX1-A. (A) Volcano plot of up- and downregulated genes in TaLAX1-A-OE transgenic calli vs. empty vector (control) transgenic calli of Chinese Spring. Purple, cytokinin-related genes; red, auxin-related genes; green, TaGRFs and TaGIF1-A. (B) Gene Ontology (GO) enrichment analysis of up- and downregulated genes from TaLAX1-A-OE transgenic calli vs. empty vector (control) transgenic calli. Red, GO terms of genes upregulated in TaLAX1-A-OE vs. control; green, GO terms of genes downregulated in TaLAX1-A-OE vs. control. (C) Relative expression levels of selected genes regulated by TaLAX1-A in TaLAX1-A-OE transgenic calli and empty vector transgenic calli. (D) ChIP–qPCR of the TaGIF1-A promoter using an anti-myc antibody in the TaLAX1-A-OE transgenic lines. TaLAX1-A-OE samples with IgG antibody were used as negative controls. TaLAX1-A binds to the P2 and P4 regions of the TaGIF1-A promoter. (E) Transient expression of TaLAX1-A protein and TaGIF1-A_pro:LUC reporter in tobacco leaves (left) and statistics of luciferase activity (right). (F) ChIP–qPCR of the TaARF3-D promoter using an anti-myc antibody in the TaLAX1-A-OE transgenic lines; TaLAX1-A-OE samples with IgG antibody were used as a negative control. TaLAX1-A binds to the P1, P2, and P7 regions of the TaARF3-D promoter. (G) Transient expression of TaLAX1-A protein and TaARF3-D_pro:LUC reporter in tobacco leaves (left) and statistics of luciferase activity (right). (H) Endogenous IAA content in TaLAX1-A-OE transgenic calli and empty vector (control) transgenic calli. Values in (C), (D), and (F) are means ± SD; values in (E), (G), and (H) are means ± SEM. All experiments were performed at least three times. Black points are the results from individual experiments. ∗∗∗∗P < 0.0001; ∗∗∗P < 0.001; ∗∗P < 0.01; ∗P < 0.05; ns, not significant (Student’s t-test, two-tailed).

Figure 6

TaLAX1-A activates cytokinin biosynthesis during the process of shoot regeneration. (A) Expression heatmap of genes related to cytokinin biosynthetic and metabolic processes in control (empty vector) transgenic calli and TaLAX1-A-OE transgenic calli. (B) ChIP–qPCR of the TaIPT1-A promoter using an anti-myc antibody in the TaLAX1-A-OE transgenic lines; TaLAX1-A-OE samples with IgG antibody were used as a negative control. TaLAX1-A binds to the P6 region of the TaIPT1-A promoter. (C) Transient expression of TaLAX1-A protein and TaIPT1-A_pro:LUC reporter in tobacco leaves (left) and statistics of luciferase activity (right). (D) Endogenous cytokinin content (IPR, TZ, TZR, CZ, CZR, and DHZR) in TaLAX1-A-OE transgenic calli and empty vector (control) transgenic calli. (E) Shoot regeneration phenotypes of Fielder embryos transformed with the empty vector (control) or TaLAX1-A-OE after incubation on CIM for 42 days and then on SIM (without exogenous cytokinin) for 20 days. Scale bar, 1 cm. (F) Regeneration frequencies and regenerating shoot frequencies of Fielder immature embryos infected with control or TaLAX1-A-OE after incubation on CIM for 42 days and then on SIM (without exogenous cytokinin) for 20 days. Values in (B) are means ± SD; values in (C), (D), and (F) are means ± SEM. All experiments in (B)–(D) and (F) were performed at least three times. Black points are the results from individual experiments. ∗∗∗∗P < 0.0001; ∗∗∗P < 0.001; ∗∗P < 0.01; ∗P < 0.05; ns, not significant (Student’s t-test, two-tailed).

Figure 7

Transformation of TaLAX1 homologs into maize and soybean. (A) Shoot regeneration phenotypes of maize (B104) transformed with the empty vector (control, p3300-CUB). Scale bar, 1 cm. (B) Shoot regeneration phenotypes of maize (B104) transformed with ZmBA1-OE. Scale bar, 1 cm. (C) Regeneration frequencies of B104 immature embryos infected with control or ZmBA1-OE. Regeneration frequency = no. of calli showing at least one regenerating shoot/no. of inoculated embryos × 100%. (D) Shoot regeneration phenotypes of soybean (Dongnong-50) transformed with the empty vector (control, 35S-PBI106). Scale bar, 1 cm. (E) Shoot regeneration phenotypes of soybean (Dongnong-50) transformed with GmLAX1-OE. Regenerated shoots are indicated with white arrows. Scale bar, 1 cm. (F) Regeneration frequencies of Dongnong-50 cotyledonary nodes infected with control or GmLAX1-OE. Regeneration frequency = no. of explants showing at least one regenerating shoot/no. of inoculated explants × 100%. Values in (C) and (F) are means ± SEM from three independent experiments. Black points are the results from individual experiments. ∗P < 0.05 (Student’s t-test, two-tailed).