Application of Wox2a in transformation of recalcitrant maize genotypes

The genetic transformation plays an important role in plant gene functional analysis and its genetic improvement. However, only a limited number of maize germplasms can be routinely transformed. The maize gene Wuschel-like homeobox protein 2a (Wox2a) was shown to play a crucial role in promoting the formation of embryonic cells and enhancing the efficiency of genetic transformation in maize. This commentary discusses the mechanism by which the Wox2a gene contributes to the variation in embryogenic tissue culture response among different maize inbred lines. In addition, the frequency and intensity of Wox2a or Wus2/Bbm vector-induced somatic embryogenesis was also discussed. The application of Wox2a in transformation of recalcitrant maize genotypes could well accelerate the development of maize genetic improvement.

Plant gene functional analysis and its genetic improvement rely mainly on genetic transformation.Maize is a critical cereal crop in global agriculture, alongside other essential crops like rice, wheat, and barley.Unfortunately, as with these crops, only a limited number of maize germplasms can be routinely transformed, thereby significantly restricting exploration of gene functions and their potential for genetic improvement.Elevating the expression levels of the maize (Zea mays) Baby Boom (Bbm) and Wuschel2 (Wus2) genes resulted in a significant increase in transformation frequencies in previously non-transformable maize inbred lines.However, some negative effects, including phenotypic abnormality and sterility, were observed in these Bbm and Wus2 overexpression lines.To obtain normal phenotypes for transgenic plants, the desiccation-inducible promoter, Rab17pro, was employed to control expression of the CRE recombinase gene.This approach successfully eliminated the undesired effects of Bbm and Wus2 overexpression (Lowe et al. 2016).Furthermore, to generate healthy and fertile plants, without any callus formation, Lowe et al. (2018) utilized two other promoters: the maize phospholipid transferase gene promoter (Zm-PLTPpro) and the auxin-inducible promoter (Zm-Axig1), to drive the expression of Bbm and Wus2, respectively (Table 1).
In a recent report by McFarland et al. (2023), the maize gene Wuschel-like homeobox protein 2a (Wox2a) was shown to play a crucial role in promoting the formation of embryonic cells and enhancing the efficiency of genetic transformation in maize.The T 0 plants were successfully obtained through the regeneration of somatic embryos derived from the explants transformed with the Wox2a-overexpressing vectors.These regenerated plants exhibited a normal phenotype and displayed full fertility (Table 1), suggesting that the Wox2a gene has significant potential for applications in the genetic transformation of maize.
How was the Wox2a gene identified?McFarland et al. (2023) selected Wox2a as the candidate gene based on a set of criteria.The Wox2a gene was located within the 3 Mb fine-mapping region on chromosome 3, which had a known association with embryogenic culture aBIOTECH response, as determined by its genetic position and expression pattern (Salvo et al. 2018).Moreover, in the immature zygotic embryos of A188, a preferred inbred line for tissue culture and transformation applications, the Wox2a transcripts exhibited an eightfold increase at 24 h after being introduced into tissues (Salvo et al. 2014).In addition, the Wox2a gene showed transcriptional activity in both the zygote and the apical domain of the early embryo in maize.Similarly, its orthologous gene, AtWox2, displayed activity in the zygote and played a crucial role in initiating the embryonic shoot apical meristem in Arabidopsis (Haecker et al. 2004;Nardmann et al. 2007;Zhang et al. 2017).This suggests that the function of Wox2a in embryo development is conserved between maize and Arabidopsis.
What is the mechanism by which the Wox2a gene contributes to the variation in embryogenic tissue culture response among different maize inbred lines?One assumption suggested that there could be differences in the sequences, or functions, of the Wox2a gene between recalcitrant and readily transformable maize genotypes B73 (an essential inbred line in maize genetics and genomics study) and A188.McFarland et al. (2023) determined that the B73 and A188 alleles of Wox2a shared only approx.69% identity in their promoter sequences, whereas the coding sequences for both genotypes displayed a high degree of conservation.
The transgenic cells, transformed with both expression vectors ZmUbi1::Wox2a-B73 and ZmUbi1::Wox2a-A188, showed similar frequencies of somatic embryo formation and plantlet regeneration, indicating that the Wox2a gene in different maize inbred lines (B73 and A188), with varying abilities to form embryonic cells, has the same function in embryogenesis and cell differentiation.According to these findings, an alternative assumption proposes that the diverse genetic transformation capacities of B73 and A188 could be attributed to variations in the expression of the Wox2a gene.McFarland et al. (2023) detected the Wox2a gene expression in maize inbred lines exhibiting varying genetic transformation abilities.They observed that the expression level of Wox2a in the highly embryogenic  and regenerable maize genotype Hi-II AxB immature embryos was 1.3-fold higher than that in the B73 embryos.This suggests that elevating the expression of the Wox2a gene, in the recalcitrant maize genotype, may be sufficient to trigger somatic embryogenesis in its immature embryos.Hence, the Wox2a gene was introduced into the immature zygotic embryos of the maize inbred line LH244, which typically exhibited a slower-growing type I embryogenic callus response, compared to the fastgrowing type II response observed in A188.The introduction of the overexpressed Wox2a gene resulted in direct somatic embryogenesis in the immature embryos of LH244.Here, some 20-22% of explants treated with Wox2a expression, under the ZmUbi1 promoter, generated somatic embryos at a level significantly higher than the control.However, the Wox2a overexpression vector induced somatic embryogenesis at a lower frequency and intensity than Wus2/Bbm (Fig. 1 A and B).
In summary, McFarland et al. (2023) identified a crucial gene, Wox2a, using a forward genetic strategy and established that it could induce somatic embryo formation and promote the growth of friable, embryogenic, and regenerable callus in the recalcitrant maize genotype B73.Of equal importance, Wox2a enabled the regeneration of phenotypically normal plants and their subsequent offspring.Integration of these findings into maize genetic breeding programs could well accelerate the development of elite lines with traits tailored for yield performance under regional-specific conditions.

Table 1
Use of Bbm, Wus and Wox2a to enhance transformation of maize pro ::Bbm plus Nos pro ::Wus2 High