Establishment of Agrobacterium-mediated Transformation System for Agropyron mongolicum

doi:10.11983/CBB24012

Abstract

Abstract: Agropyron mongolicum is a perennial shrub grass in the Triticeae tribe of Poaceae family. It has high forage quality and tolerance to cold, drought, salt and sandstorm. Therefore, it is an excellent grass species for mining stress tolerant genes and restoring deteriorated grasslands. However, a highly efficient transformation system has yet to be established in A. mongolicum. It restricts the identification of genetic resources and the application of genetic improvement of this species. Here, we report an embryogenic callus line (ECL) #89 that was induced from the seeds of A. mongolicum cultivar Mengnong No. 1. Our studies showed the ECL #89 had high regeneration and Agrobacterium-infection efficiencies. An Agrobacterium-mediated transformation system with a transformation efficiency up to 30% was established by infecting the ECL #89 with EHA105. Additionally, we found that subcultures could decrease severely the regeneration capacity of ECL #89, which could be partially restored by adding (1 mg·L^-1) ABA or high concentration (45 g·L^-1) sucrose in the regeneration medium, from 5% to 35% and 42%, respectively. Our work will facilitate the developing of genome editing system, gene function characterization, and molecular breeding in A. mongolicum.

Key words: Agropyron mongolicum, embryogenic callus, Agrobacterium tumefaciens, genetic transformation

Yuchen Li, Haixia Zhao, Xiping Jiang, Xintian Huang, Yaling Liu, Zhenying Wu, Yan Zhao, Chunxiang Fu. Establishment of Agrobacterium-mediated Transformation System for Agropyron mongolicum[J]. Chinese Bulletin of Botany, 2024, 59(4): 600-612.

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URL: https://www.chinbullbotany.com/EN/10.11983/CBB24012

https://www.chinbullbotany.com/EN/Y2024/V59/I4/600

Figures/Tables 7

Figure 1 Schematic diagram of pANIC6B empty vector (refer to Mann et al., 2012) LB: Left border of T-DNA; OsAct1: Rice Act1 promoter and intron; hph: Screening marker gene for hygromycin resistance; 35S T: 35S terminator; PvUbi1: Switchgrass Ubi1 promoter and intron; GUSplus: β-glucuronidase plus reporter gene; NOS T: Agrobacterium tumefaciens carmine synthase gene terminator; ZmUbi1: Maize Ubi1 promoter and intron; Gateway cassette: attR1-CmR/ccdb-attR2; OCS T: Octopine synthase terminator; RB: Right border of T-DNA

Figure 2 Phenotypes of callus lines induced from Mengnong No.1 seeds (A)-(D) Phenotypes of different callus lines (the up photos are calli grown on M5 medium, the down photos are calli under the microscope. Bars=1 cm); (E) Percentage of embryogenic callus lines (ECL) in total callus lines (One-way analysis of variance, Duncan’s test, P<0.05).

Figure 3 Regeneration analyses of embryogenic callus lines (ECL) induced from Mengnong No.1 seeds (A) Regeneration of embryogenic callus lines #88 and #89 with different genotypes (bars=1 cm); (B) Percentage of embryogenic callus lines with more than 30% regeneration efficiency in total embryogenic callus lines. Different lowercase letters indicate significant differences among three experimental groups (One-way analysis of variance, Duncan’s test, P<0.05).

Figure 4 Effects of ABA and high concentration sucrose on the regeneration capacity of deteriorated embryogenic callus line #89 (A) Differentiation of deteriorated embryogenic callus line #89 grown in MSBK medium; (B) Differentiation of deteriorated embryogenic callus line #89 grown in MSBKA medium supplemented with 1 mg·L-1 ABA; (C) Differentiation of deteriorated embryogenic callus line #89 grown in MSBKS45 medium supplemented with 45 g·L-1 sucrose; (D) Regeneration efficiency of deteriorated embryogenic callus line #89 grown in MSBKA medium with ABA treatment; (E) Regeneration efficiency of deteriorated embryogenic callus line #89 grown in MSBKS45 medium with 30 g·L-1 and 45 g·L-1 sucrose. Different lowercase letters indicate significant differences between treatments (One-way analysis of variance, Duncan’s test, P<0.05). The callus depicted in (A), (B), and (C) on the right side represents the microscopic observation of the corresponding structures on the left side. Bars=1 cm

Figure 5 Infection analyses of embryogenic callus lines (ECL) induced from Mengnong No.1 seeds (A) The infection efficiency of EHA105 on embryogenic callus lines through GUS staining; (B) The percentage of embryogenic callus lines with high infection efficiency (>40%) in total embryogenic callus lines. Different lowercase letters indicated significant differences among three experimental groups (One-way analysis of variance, Duncan’s test, P<0.05).

Figure 6 Establishment of the Agrobacterium-mediated transformation in Mengnong No.1 (A) #89 embryogenic callus line induced from Mengnong No.1 seeds grown on M2 medium; (B) Co-cultivation of #89 calli with Agrobacterium strain EHA105; (C) Agrobacterium infection analysis of #89 embryogenic calli by GUS staining; (D) Selection of hygromycin resistant calli through M2H30 medium; (E) GUS staining of hygromycin resistant callus; (F) Differentiation of the hygromycin resistant calli on MSBKH2 medium; (G) Growth of the hygromycin resistant plant in soil; (H) GUS staining of leaves from the hygromycin resistant plants. Bars=1 cm

Figure 7 PCR analysis of hph in Mengnong No.1 Agropyron mongolicum transgenic plants M: 2000 bp DNA marker; 1-19: PCR template (1: pANIC6B empty vector; 2: ddH2O; 3: Non-transgenic plant regenerated from embryogenic callus line #89; 4-19: Transgenic plants regenerated from embryogenic callus #89. The sizes of PCR products are 375 bp for hph).

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