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Breeding exceptionally fragrant soybeans for soy milk with strong aroma
Monday, 2024/03/04 | 08:39:25
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Hongtao Xie, Minglei Song, Xuesong Cao, Qingfeng Niu, Jianhua Zhu, Shasha Li, Xin Wang, Xiaomu Niu, Jian-Kang Zhu Journal of Integrative Plant Biology; First published: 23 February 2024; https://doi.org/10.1111/jipb.13631
Soybean (Glycine max (L.) Merr.) is a major source of vege-table protein and oil in human diet and animal nutrition.Soybean seeds have been extensively used in various foodproducts and snacks. Taste quality, particularly the aroma, af-fects cooking and eating, and ultimately influences consumerpreference. Soy milk is particularly popular in China and hasbeen gaining popularity in many other countries in the world.
In our experiment, we utilized the GmEF1α promoter to drive the expression of Cas12i3 (Duan et al., 2024). We employed the GmU6 promoter to drive crRNA1 and the GmU3 promoter to drive crRNA2 (Figure 1A), which target the fourth exons of GmBADH1 and GmBADH2, respectively (Figure 1B, C). To assess the editing efficiency of Cas12i3 in soybean, we employed a hairy root test system, as previously described (Cao et al., 2022). We analyzed the GmBADH1 and GmBADH2 mutations in hairy roots by Sanger sequencing. In 20 transformed hairy roots examined, mutation frequencies were found to be 45.0% for GmBADH1 and 10.0% for GmBADH2 (Figure 1D). This result showed that the CRISPR/Cas12i3 gene editing system is active in soybean. Following the successful hairy roots experiment, the binary vector was introduced into XD20 through Agrobacterium tumefaciens-mediated stable transformation as previously described (Paz et al., 2006). As a result, we obtained 103 independent T0 transgenic lines, 24 of which were found to have mutations in either or both of the target genes (Figure 1E). Soybean is widely used to produce soy milk due to its pleasant taste and health benefits. As anticipated, soy milk made from the gmbadh1/2 double mutant seeds exhibited a much stronger aromatic smell that was consistent with its much higher 2-AP level (Figure 1U). This heightened aroma likely enhances the sensory experience of consuming soy milk, which is supported by the preferences of volunteers when given the choice between soy milk made from nonaromatic soybeans and that produced from the gmbadh1/2 soybean seeds (Figure 1V).
See more https://onlinelibrary.wiley.com/doi/10.1111/jipb.13631
Figure 1: CRISPR/Cas12i3-mediated editing of GmBADH1 and GmBADH2 in soybean enhances 2-AP content and aroma (A) Schematic of the CRISPR/Cas12i3 construct. (B) GmBADH1 crRNA design and mutant genotypes. (C) GmBADH2 crRNA design and mutant genotypes. (D, E) Editing efficiency in hair roots and T0 mutants. (F) Double mutant genotypes. (G) Photograph of XD20 at 16 DAE (days after emergence). (H) Photograph of gmbadh1-1 (left) and gmbadh1-2 (right) at 16 DAE. (I) Photograph of gmbadh2-1 (left) and gmbadh2-2 (right) at 16 DAE. (J) Photograph of gmbadh1/2 double mutant #3 (left) and #6 (right) at 16 DAE. (K) Photograph of XD20 at 74 DAE. (L) Photograph of gmbadh1/2 double mutant #3 (left) and #6 (right) at 74 DAE. (M) Photograph of XD20 at 98 DAE. (N) Photograph of gmbadh1/2 double mutant #3 (left) and #6 (right) at 98 DAE. (O–R) The plant height (O), flowering time (P), seeds weight (Q) and maturity time (R) of XD20 and the mutants. (S–U) The 2-AP content of dry leaves (S), dry seeds (T) and soybean milk (U) of XD20 and the mutants. (V) The number of volunteers choosing XD20 or the double mutant soy milk. Data (O–V) are shown as the means ± SE (n = 3). P-values were calculated using a t-test (V) or ANOVA (R–U). *P < 0.05. ****P < 0.0001. Red bases are target sequences and the underlined bases represent the protospacer adjacent motif (5′-TTN). Bars = 14 cm.
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