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Acta Agronomica Sinica ›› 2023, Vol. 49 ›› Issue (7): 1758-1768.doi: 10.3724/SP.J.1006.2023.24197

• CROP GENETICS & BREEDING·GERMPLASM RESOURCES·MOLECULAR GENETICS • Previous Articles     Next Articles

Establishment and optimization of a high-efficient hairy-root system in foxtail millet (Setaria italica L.)

WAN Yi-Man(), XIAO Sheng-Hui, BAI Yi-Chao, FAN Jia-Yin, WANG Yan, WU Chang-Ai*()   

  1. School of Life Science, Shandong Agricultural University, Tai’an 271000, Shandong, China
  • Received:2022-08-26 Accepted:2022-11-25 Online:2023-07-12 Published:2022-12-06
  • Contact: *E-mail: cawu@sdau.edu.cn E-mail:wanyiman123@163.com;cawu@sdau.edu.cn
  • Supported by:
    The National Key Research and Development Program of China(2018YFD1000704);The National Key Research and Development Program of China(2018YFD100070);The Key Research and Development Program (Agricultural Fine Seed Project) of Shandong Province(2021LZGC006)

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Abstract:

To establish a system for rapid identification of millet gene functions, in this study, we compared the effects of explants from different foxtail millet cultivars, the concentrations of acetyleugenone, bacterial solution, and co-culture time on the hairy root induction mediated by Agrobacteria rhizogenes strain K599. When the shoot tips were infected with bacterial solution 0.5 optical density (OD) and containing 100 μmol L-1 acetyleugenone, and co-cultured for three days, the induction efficiency of hairy roots reached to 80.24%. Using this system, we analyzed the transformation efficiency of the hairy roots, the subcellular localization of SiDVL1and SiDVL3, and gene functions of SiNHX2, SiCBL4, and SiCBL7. The PCR results of GFP gene and GFP fluorescence microscopic observation indicated that the transformation efficiency of the hairy roots was over 70%. The subcellular localization of SiDVL1 and SiDVL3 was similar in millet hairy-root and tobacco epidermal cells. Simultaneously, the survival rates of SiNHX2, SiCBL4, and SiCBL7 transgenic millet were significantly higher than that of empty vector transformed millet. Therefore, our study established an efficient and rapid method to analyze the subcellular location and to identify the function of genes from foxtail millet.

Key words: Agrobacterium rhizogenes, hairy roots, foxtail millet (Setaria italica), transformation efficiency, gene function identification

Fig. 1

Effects of different types of explants on hairy roots induction A and B: Foxtail millet’s hairy-root development after A. rhizogenes strain K599 infecting embryogenic callus for 10 days and bud tips for 5 days, respectively. C: the root length of induced hairy roots cultured for 14 days. D: the induction efficiency of hairy roots cultured for 14 days. Three biological experiments were repeated, n = 30. One-way ANOVA Duncan’s test is used for statistical analysis. Different letters indicate significant differences at P < 0.05."

Fig. 2

Effects of bud tip explants from different millet varieties on hairy roots induction A-E: the effects of Ci846, xiaomi, Dungu, Longzhu 1, and Yugu 1 on hairy roots induction, respectively. F: the root length of induced hairy roots using bud tips from indicated varieties as the explants. G: the induction efficiency of hairy roots when bud tips from indicated varieties were used as the explants. H: the average induced hairy root number when bud tips from indicated varieties were used as the explants. Three biological experiments were repeated, n = 30. One-way ANOVA Duncan’s test is used for statistical analysis; ns indicates no significant correlation at the 0.05 probability level."

Fig. 3

Effects of AS on foxtail millet hairy roots induction A-E: the effect of 0, 50, 100, 150, and 200 μmol L-1 AS on hairy roots induction, respectively. F: the root length of induced hairy roots in the presence of 0, 50, 100, 150, and 200 μmol L-1 AS. G: the induction efficiency of hairy roots in the presence of 0, 50, 100, 150, and 200 μmol L-1 AS. H: the average induced root number in the presence of 0, 50, 100, 150, and 200 μmol L-1 AS. Three biological experiments were repeated, n = 30. One-way ANOVA Duncan’s test is used for statistical analysis. Different letters indicate significant differences at P < 0.05."

Fig. 4

Effect of bacterium concentration on foxtail millet hairy roots induction A-F: the effect of bacteria solution at concentrations 0.1, 0.2, 0.3, 0.5, 0.8, and 1.0 OD600 on hairy roots induction, respectively. G: the root length of induced hairy roots by bacteria at concentrations mentioned in (A-F), respectively. H: the induction efficiency of hairy roots by bacteria solution at concentrations mentioned in (A-F), respectively. I: the average induced rooting number by bacteria solution at concentrations mentioned in (A-F), respectively. Three biological experiments were repeated, n = 30. One-way ANOVA Duncan’s test is used for statistical analysis. Different letters indicate significant differences at P < 0.05."

Fig. 5

Effect of co-culture time on foxtail millet hairy roots induction A-E: the effects of co-culture time of 1, 2, 3, 4, and 5 day(s) on hairy roots induction, respectively. F: the root length of induced hairy roots after co-culture for 1, 2, 3, 4, and 5 day(s). G: the induction efficiency of hairy roots after co-culture for 1, 2, 3, 4, and 5 day(s). H: the average hairy root number after co-culture for 1, 2, 3, 4, and 5 day(s). Three biological experiments were repeated, n = 30. One-way ANOVA Duncan’s test is used for statistical analysis. Different letters indicate significant differences at P < 0.05."

Table 1

Data statistics of hairy root by shoot tip explants induction"

试验编号
Test ID		 侵染的芽尖总数
Total number of
infected shoot tips		 诱导发根的芽尖数
Number of bud tips to induce rooting		 发根总数
Total bud tip rooting number		 平均发根数
Average rooting number		 发根率
Rooting rate
(%)
1 19 17 63 4 89.47
2 22 17 44 3 77.27
3 21 14 44 3 66.67
4 21 21 65 3 100.00
5 21 17 64 2 80.95
6 25 19 57 3 76.00
7 20 17 55 3 85.00
8 22 18 66 3 81.82
9 18 14 41 5 77.78
10 21 17 50 3 80.95
11 20 13 29 2 65.00
12 23 19 38 2 82.61
总计 Total 253 203 616 3 80.24

Fig. 6

Verification of transgenic hairy root by PCR M: DNA marker. 1: the gene expression vector was used as the positive control. 2-33: DNA from induced hairy root samples. WT: the empty vector from induced hairy root samples was used as the negative control."

Fig. 7

Subcellular localization of SiDVL genes using A. rhizogenes-mediated hairy root transformation"

Fig. 8

Responses of foxtail millet with SiNHX2, SiCBL4, and SiCBL7 transformed hairy-roots to salt stress A: the phenotypes of millet seedlings with SiNHX2, SiCBL4, and SiCBL7 transformed hairy-roots when treated on MS medium with or without 175 mmol L-1 NaCl for 7 days. B: the survival rates of millet seedlings with SiNHX2, SiCBL4, and SiCBL7 transformed hairy-roots when treated on MS medium with or without 175 mmol L-1 NaCl for 7 days. Three biological experiments were repeated, n = 30. ns indicates no significant correlation in the 0.05 probability level; *** indicates significant correlation at the 0.01 probability level."

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