Establishment and Optimization of a Shoot Tip-based Genetic Transformation System for Foxtail Millet

doi:10.11983/CBB20119

Abstract

Abstract:

In this study, a simple and stable genetic transformation system of foxtail millet (Seteria italica) was established and optimized, in which shoot tips were used as the explant. We transformed Yugu 1, an elite millet cultivar, by Agrobacterium-mediated transformation, and tested different treatments to boost transformation efficiency. We used a PCR-based assay to screen transformants in third-leaf stage seedlings. We determined an optimal lethal concentration of glufosinate (Basta) when sprayed to millet seedlings, and tested the different PCR-based genotyping methods with or without Basta spary. Using the newly established pipeline, we further optimized various crucial factors that affect genetic transformation efficiency. We found that an optimal concentration of bacterial culture was OD₆₀₀=1.4, an optimal concentration of acetolsyringone was 800 μmol∙L ^-1. We also obtained high transformation efficiency with an infecting pressure at 0.05 MPa, and an infecting time of 40 min. We used the above-mentioned transformation method to transform a Seteria italica calcineurin B-like protein 4 (SiCBL4) overexpression construct. Genetic stability analysis on T₂ generation transformed plants was performed by the combination assay of Basta resistance and real-time quantitative fluorescence RT-PCR, which can save the time of genotyping. Altogether, this study establishes a shoot tip-based stable genetic transformation system for foxtail millets, and also develops a robust pipeline to detect transgenic offsprings.

Key words: foxtail millet, genetic transformation, glufosinate, in vitro, shoot tip

Lan Yang, Ya Liu, Yang Xiang, Xiujuan Sun, Jingwei Yan, Aying Zhang. Establishment and Optimization of a Shoot Tip-based Genetic Transformation System for Foxtail Millet[J]. Chinese Bulletin of Botany, 2021, 56(1): 71-79.

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URL: https://www.chinbullbotany.com/EN/10.11983/CBB20119

https://www.chinbullbotany.com/EN/Y2021/V56/I1/71

Figures/Tables 8

Figure 1 The T-DNA region of plasmid pCUN-NHF LB: LB T-DNA repeat; Ubi promoter: Zea mays Ubiquitin1 promoter; HA: HA tag; MCS: Multiple cloning site; 35S promoter: CaMV35S promoter; Bar: Herbicide bialaphos-resistance gene; RB: RB T-DNA repeat

Figure 2 Identification of transgenic foxtail millet (A) Image from GUS staining of foxtail millet seedlings (bar=0.1 cm); (B) PCR identification (1: Positive control; 2: Negative control; 3: A leaf with one wound cut; 4: A leaf with two wounds cut; 5: A leaf without wound)

Figure 3 Establishment of the genetic transformation system for foxtail millet using shoot tip (A) Seeds to be germinated; (B) Seeds after germination for 3 d; (C) Seedlings after transformation for 5 d; (D) Plants at jointing stage; (E) Plants at heading stage; (F) Mature seeds. (A), (C), (E) Bars=1.0 cm; (B) Bar=0.5 cm; (D) Bar=10.0 cm; (F) Bar=5.0 cm

Figure 4 Effects of glufosinate concentrations on survival rate of foxtail millet seedlings (A) Phenotypes of foxtail millet seedlings treated with glufosinate at different concentrations (bar=5.0 cm); (B) The survival rate of foxtail millet seedlings treated with different concentrations of glufosinate. Data are means±SD (n=3). Student’s t-test, ** P<0.01, *** P<0.001

Figure 5 Transformation rate obtained using two different methods Data are means±SD (n=3). Student’s t-test, * P<0.05

Figure 6 Effects of Agrobacterium concentration (A), acetosyringone concentration (B), infecting pressure (C), infecting time (D) and changing four variables (E) on transformation rates for foxtail millet using shoot tip Data are means±SD (n=3). Student’s t-test, * P<0.05. Different lowercase letters indicat significant differences (P<0.05).

Table 1 Comparison of the conditions of the initially established system and the optimized system

	Agrobacterium concentration (OD₆₀₀)	Acetosyringone concentration (μmol?L^-1)	Pressure (MPa)	Time (min)
Before optimization	1.0	400	0.04	20
After optimization	1.4	800	0.05	40

Figure 7 qRT-PCR analysis of the expression of SiCBL4 in transgenic foxtail millet Data are means±SD (n=3). Student’s t-test, * P<0.05, ** P<0.01

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