Chinese Bulletin of Botany ›› 2021, Vol. 56 ›› Issue (1): 62-70.DOI: 10.11983/CBB20174
• TECHNIQUES AND METHODS • Previous Articles Next Articles
Pengfei Du1,2,3, Yu Wang2, Yingping Cao2, Song Yang2, Zhichao Sun2, Decai Mao3,4, Jiajun Yan3,4, Daxu Li3,4, Meizhen Sun5, Chunxiang Fu2,*(), Shiqie Bai3,4,*(
)
Received:
2020-11-02
Accepted:
2021-01-05
Online:
2021-01-01
Published:
2021-01-15
Contact:
Chunxiang Fu,Shiqie Bai
Pengfei Du, Yu Wang, Yingping Cao, Song Yang, Zhichao Sun, Decai Mao, Jiajun Yan, Daxu Li, Meizhen Sun, Chunxiang Fu, Shiqie Bai. Establishment of Biolistic Mediated Transformation System for Elymus sibiricus[J]. Chinese Bulletin of Botany, 2021, 56(1): 62-70.
Figure 1 T-DNA segment of PANIC6A (A), PANIC6D (B) and PANIC6E (C) LB: Left boundary of T-DNA segment; OsAct1: Rice promoter; hph/bar: Screening marker genes; PvUbi1: Switchgrass promoter; pporRFP/GUSplus: Red fluorescence/glucuronidase reporter gene; ZmUbi1: Maize promoter; Cmr: Chloramphenicol resistant gene; ccdB: Target gene; RB: Right boundary of T-DNA segment
Infect schemes | Infection methods | |||
---|---|---|---|---|
Predrying treatment (10 min) | Vacuum treatment (10 min) | Ultrasonic treatment (5 min) | Vacuum treatment (10 min) | |
1 | - | + | + | + |
2 | - | + | + | - |
3 | - | - | + | + |
4 | + | + | + | + |
Table 1 Four Agrobacterium infection methods
Infect schemes | Infection methods | |||
---|---|---|---|---|
Predrying treatment (10 min) | Vacuum treatment (10 min) | Ultrasonic treatment (5 min) | Vacuum treatment (10 min) | |
1 | - | + | + | + |
2 | - | + | + | - |
3 | - | - | + | + |
4 | + | + | + | + |
Figure 2 Micrographs of calli from different explants (30 d) (A)-(E) Callus induced by seeds (mature embryo), roots, hypocotyl, stem and inflorescence, respectively. Bars=50 μm
Figure 3 Callus induction time and induction rate of different explants in Elymus sibiricus cv. ‘Chuancao No.2’ The error bars indicate standard error (SE), the sample size was 60. Different lowercase letters indicate significant differences (P<0.05).
Figure 4 The process diagram of tissue culture and regeneration system and the plot of callus differentiation efficiency of Elymus sibiricus cv. ‘Chuancao No.2’ with subculture time (A) Inflorescence of ‘Chuancao No.2’ placed in callus induction medium; (B) Callus induced by inflorescence of ‘Chuancao No.2’ for 35 d; (C) The callus with dense and hard structure in figure (B) was selected and placed in medium for 15 d; (D) Callus in figure (C) cultured in differentiation medium for 35 d; (E) The micrograph of callus in red frame in figure (D); (F) Differentiated seedlings in figure (D) placed in rooting medium for 30 d; (G) Seeds placed in callus induction medium; (H) Callus induced by seeds for 35 d; (I) The callus with dense and hard structure in figure (H) was selected and placed in subculture medium for 15 d; (J) Callus in figure (I) cultured in differentiation medium for 35 d; (K) The micrograph of callus in red frame in figure (J); (L) The differentiation efficiency of callus induced by inflorescence at different time periods (The error line is the standard error, and the sample size is 30; Different lowercase letters indicate significant differences at P<0.05). Bars in (D), (F), (G), (J)=2 cm; Bars in (A)-(C), (E), (H), (I), (K)=1 cm
Figure 5 Callus of inflorescence and inflorescence infected by Agrobacterium tumefaciens in Elymus sibiricus cv. ‘Chuancao No.2’ (A), (B) Callus induced by inflorescence before and after GUS staining after Agrobacterium infection; (C), (D) The bright and dark field images of RFP fluorescence after Agrobacterium infection of callus induced by inflorescence; (E), (F) The images before and after GUS staining directly after Agrobacterium infection with inflorescence; (G), (H) The bright and dark field images of RFP fluorescence after Agrobacterium infection of inflorescence, respectively. Bars=50 μm
Figure 6 Fluorescence micrographs of inflorescence callus after bombardment with different induction time and pretreatment methods (A), (B) Callus of inflorescence on 25 d (no bombardment); (C), (D) Conventional hyperosmotic treatment of inflorescence callus at 25 d; (E), (F) Inflorescence callus filter paper dried for 2 h on 25 d; (G), (H) Inflorescence callus filter paper dried for 2 h on 35 d. Arrows indicate the positive transgenic calli. Bars =20 μm
Figure 7 The trend of percentage of fluorescent callus after gene gun bombardment with continuous observation time The error line is the standard error, and the sample size is 30. The fluorescence callus ratio of the 25-day inflorescence callus after 2 h drying on filter paper was significantly higher than that of the other two treatments (* P<0.05, ** P<0.01, *** P<0.001).
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