Establishment of Biolistic Mediated Transformation System for Elymus sibiricus

doi:10.11983/CBB20174

Abstract

Abstract: Elymus sibiricus cv. ‘Chuancao No.2’ is the main cultivated grass species for desertification control and construction of high-yield and high-quality pasture in northwest Sichuan Plateau. In this study, we tested five explants of E. sibiricus cv. ‘Chuancao No.2’ for callus induction, and found that only inflorescence calli were able to differentiate and regenerate. The calli of inflorescence with dense and hard structure cultured for 25 d and 35 d were used for Agrobacterium and biolistic mediated transformation respectively. The results showed that only biolistic-mediated transformation could produce positive transgenic calli of ‘Chuancao No.2’. In the process of biolistic-mediated transformation, the calli was pretreated in two ways: hyperosmotic culture and filter paper drying. The results revealed that the transformation efficiency of filter paper drying was higher than that of hyperosmotic treatment. For the inflorescence callus after 25 d induction, the transformation efficiency under the condition of 2 h drying of filter paper was highest which reached about 40%. In short, we applied the biolistic technology in ‘Chuancao No.2’ for the first time and successfully obtained the positive transgenic inflorescence calli. This work will lead to establishment of the robust transformation system for E. sibiricus in future.

Key words: Elymus sibiricus cv. ‘Chuancao No.2’, inflorescence, Agrobacterium infection, biolistic mediated transformation, callus treatment

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.

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URL: https://www.chinbullbotany.com/EN/10.11983/CBB20174

https://www.chinbullbotany.com/EN/Y2021/V56/I1/62

Figures/Tables 8

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

Table 1 Four Agrobacterium infection methods

Infect schemes	Infection methods
Infect schemes	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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