Biotechnology Bulletin ›› 2025, Vol. 41 ›› Issue (10): 164-174.doi: 10.13560/j.cnki.biotech.bull.1985.2025-0297
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ZHANG Huan-huan1,2(
), MU Xiao-ya2, ZHOU Jing-yi2, LYU Gao-pei2, XIAO Nan2, LI Min3, HAO Yao-shan1, WU Shen-jie1,2()
Received:2025-03-19
Online:2025-10-26
Published:2025-10-28
Contact:
WU Shen-jie
E-mail:frank.red@163.com;sj__wu@126.com
ZHANG Huan-huan, MU Xiao-ya, ZHOU Jing-yi, LYU Gao-pei, XIAO Nan, LI Min, HAO Yao-shan, WU Shen-jie. An Efficient Genetic Transformation System for High-frequency Embryogenic Broomcorn Millet Line[J]. Biotechnology Bulletin, 2025, 41(10): 164-174.
| 编号 No. | 名称 Name | 编号 No. | 名称 Name |
|---|---|---|---|
| M1 | 晋黍5号 | M21 | 赤黍3号 |
| M2 | 晋黍6号 | M22 | 品糜3号 |
| M3 | 冀黍3号 | M23 | 陇糜5号 |
| M4 | 陇糜6号 | M24 | 宁糜16号 |
| M5 | 赤黍1号 | M25 | 晋黍2号 |
| M6 | 赤黍2号 | M26 | 伊糜5号 |
| M7 | 赤黍9号 | M27 | 品糜2号 |
| M8 | 宁糜10号 | M28 | 陇糜5号 |
| M9 | 陇糜8号 | M29 | 宁糜12号 |
| M10 | 固糜20号 | M30 | 宁糜15号 |
| M11 | 陇糜11号 | M31 | 赤黍5号 |
| M12 | 品糜1号 | M32 | 赤糜1号 |
| M13 | 年丰5号 | M33 | 红黍子3号 |
| M14 | 雁7号 | M34 | 黑黍子4号 |
| M15 | 齐黍1号 | M35 | 大黄糜子6号 |
| M16 | 陇糜15号 | M36 | 大黄糜子2号 |
| M17 | 宁糜13号 | M37 | 大黄糜子7号 |
| M18 | 宁糜11号 | M38 | 大黄糜子8号 |
| M19 | 赤黍8号 | M39 | 南燕川穄子2号 |
| M20 | 固糜21号 |
Table 1 39 broomcorn millet varieties in this study
| 编号 No. | 名称 Name | 编号 No. | 名称 Name |
|---|---|---|---|
| M1 | 晋黍5号 | M21 | 赤黍3号 |
| M2 | 晋黍6号 | M22 | 品糜3号 |
| M3 | 冀黍3号 | M23 | 陇糜5号 |
| M4 | 陇糜6号 | M24 | 宁糜16号 |
| M5 | 赤黍1号 | M25 | 晋黍2号 |
| M6 | 赤黍2号 | M26 | 伊糜5号 |
| M7 | 赤黍9号 | M27 | 品糜2号 |
| M8 | 宁糜10号 | M28 | 陇糜5号 |
| M9 | 陇糜8号 | M29 | 宁糜12号 |
| M10 | 固糜20号 | M30 | 宁糜15号 |
| M11 | 陇糜11号 | M31 | 赤黍5号 |
| M12 | 品糜1号 | M32 | 赤糜1号 |
| M13 | 年丰5号 | M33 | 红黍子3号 |
| M14 | 雁7号 | M34 | 黑黍子4号 |
| M15 | 齐黍1号 | M35 | 大黄糜子6号 |
| M16 | 陇糜15号 | M36 | 大黄糜子2号 |
| M17 | 宁糜13号 | M37 | 大黄糜子7号 |
| M18 | 宁糜11号 | M38 | 大黄糜子8号 |
| M19 | 赤黍8号 | M39 | 南燕川穄子2号 |
| M20 | 固糜21号 |
培养基名称 Medium name | 培养基配方 Medium composition |
|---|---|
OS培养基［ OS medium | N6盐2.5 g/L，蔗糖30 g/L，L-脯氨酸3 g/L，2，4-D 2.5 mg/L，植物凝胶2.6 g/L，pH 5.8 N6 salts 2.5 g/L， sucrose 30 g/L， L-proline 3 g/L， 2，4-D 2.5 mg/L， and phytagel 2.6 g/L， pH 5.8 |
TA培养基［ TA medium | MS盐4.4 g/L，蔗糖30 g/L，MES 500 mg/L，麦草畏2 mg/L，植物凝胶2.4 g/L，pH 5.8 MS salts 4.4 g/L， sucrose 30 g/L， MES 500 mg/L， dicamba 2 mg/L， and phytagel 2.6 g/L， pH 5.8 |
SV培养基［ SV medium | MS盐4.4 g/L，蔗糖30 g/L，五水硫酸铜 0.6 mg/L，七水硫酸锌 35 mg/L，2，4-D 2.0 mg/L，KT 0.5 mg/L，植物凝胶 2.6 g/L，pH 5.8 MS salts 4.4 g/L， sucrose 30 g/L， CuSO4·5H2O 0.6 mg/L， ZnSO4·7H2O 35 mg/L， 2，4-D 2.0 mg/L， KT 0.5 mg/L， and phytagel 2.6 g/L， pH 5.8 |
SB培养基［ SB medium | Ms盐 4.4 g/L，蔗糖30 g/L，五水硫酸铜0.16 mg/L，L-脯氨酸1 g/L，磷酸二氢钾 0.16 mg/L，2，4-D 1.0 mg/L，KT 0.5 mg/L，植物凝胶3 g/L，pH 5.8 MS salts 4.4 g/L， sucrose 30 g/L， CuSO4·5H2O 0.16 mg/L， L-proline 1 g/L， KH2PO4 0.16 mg/L， 2，4-D 1.0 mg/L， KT 0.5 mg/L， and phytagel 2.6 g/L， pH 5.8 |
SI培养基［ SI medium | MS盐4.4 g/L，蔗糖30 g/L，酸水解酪蛋白 300 mg/L， D-生物素1 mg/L， 五水硫酸铜0.6 mg/L，2，4-D 2.0 mg/L，KT 0.5 mg/L，硝酸银 2 mg/L，柠檬酸 1 mg/L，植物凝胶2.6 g/L，pH 5.8 MS salts 4.4 g/L， sucrose 30 g/L， casein hydrolysate 300 mg/L， D-biotin 1 mg/L， CuSO4·5H2O 0.6 mg/L， 2，4-D 2.0 mg/L， KT 0.5 mg/L， AgNO3 2 mg/L， citric acid 1 mg/L， and phytagel 2.6 g/L， pH 5.8 |
ZM培养基［ ZM medium | N6盐4.0 g/L，蔗糖30 g/L，2，4-D 2.0 mg/L，6-BA 0.2 mg/L，植物凝胶 2.6 g/L，pH 5.8 N6 salts 4.0g/L， sucrose 30 g/L， 2，4-D 2.0 mg/L， 6-BA 0.2 mg/L， and phytagel 2.6 g/L， pH 5.8 |
MRS培养基 MRS medium | MS盐4.4 g/L，酸水解酪蛋白300 mg/L，蔗糖30 g/L，L-谷氨酰胺100 mg/L，L-天冬酰胺100 mg/L，6-BA 1 mg/L，植物凝胶2.6 g/L，pH 5.8 MS salts 4.4 g/L， casein hydrolysate 300 mg/L， sucrose 30 g/L， L-glutamine 100 mg/L， L-asparagine 100 mg/L， 6-BA 1.0 mg/L， and phytagel 2.6 g/L， pH 5.8 |
RIM培养基 RIM medium | MS盐2.2 g/L，蔗糖30 g/L，硫酸镁0.3 g/L，IBA 1 mg/L，植物凝胶2.6 g/L，pH 5.8 MS salts 2.2 g/L， sucrose 30 g/L， MgSO4·5H2O 0.3 g/L， IBA 1.0 mg/L， and phytagel 2.6 g/L， pH 5.8 |
Table 2 Medium composition for broomcorn millet tissue culture
培养基名称 Medium name | 培养基配方 Medium composition |
|---|---|
OS培养基［ OS medium | N6盐2.5 g/L，蔗糖30 g/L，L-脯氨酸3 g/L，2，4-D 2.5 mg/L，植物凝胶2.6 g/L，pH 5.8 N6 salts 2.5 g/L， sucrose 30 g/L， L-proline 3 g/L， 2，4-D 2.5 mg/L， and phytagel 2.6 g/L， pH 5.8 |
TA培养基［ TA medium | MS盐4.4 g/L，蔗糖30 g/L，MES 500 mg/L，麦草畏2 mg/L，植物凝胶2.4 g/L，pH 5.8 MS salts 4.4 g/L， sucrose 30 g/L， MES 500 mg/L， dicamba 2 mg/L， and phytagel 2.6 g/L， pH 5.8 |
SV培养基［ SV medium | MS盐4.4 g/L，蔗糖30 g/L，五水硫酸铜 0.6 mg/L，七水硫酸锌 35 mg/L，2，4-D 2.0 mg/L，KT 0.5 mg/L，植物凝胶 2.6 g/L，pH 5.8 MS salts 4.4 g/L， sucrose 30 g/L， CuSO4·5H2O 0.6 mg/L， ZnSO4·7H2O 35 mg/L， 2，4-D 2.0 mg/L， KT 0.5 mg/L， and phytagel 2.6 g/L， pH 5.8 |
SB培养基［ SB medium | Ms盐 4.4 g/L，蔗糖30 g/L，五水硫酸铜0.16 mg/L，L-脯氨酸1 g/L，磷酸二氢钾 0.16 mg/L，2，4-D 1.0 mg/L，KT 0.5 mg/L，植物凝胶3 g/L，pH 5.8 MS salts 4.4 g/L， sucrose 30 g/L， CuSO4·5H2O 0.16 mg/L， L-proline 1 g/L， KH2PO4 0.16 mg/L， 2，4-D 1.0 mg/L， KT 0.5 mg/L， and phytagel 2.6 g/L， pH 5.8 |
SI培养基［ SI medium | MS盐4.4 g/L，蔗糖30 g/L，酸水解酪蛋白 300 mg/L， D-生物素1 mg/L， 五水硫酸铜0.6 mg/L，2，4-D 2.0 mg/L，KT 0.5 mg/L，硝酸银 2 mg/L，柠檬酸 1 mg/L，植物凝胶2.6 g/L，pH 5.8 MS salts 4.4 g/L， sucrose 30 g/L， casein hydrolysate 300 mg/L， D-biotin 1 mg/L， CuSO4·5H2O 0.6 mg/L， 2，4-D 2.0 mg/L， KT 0.5 mg/L， AgNO3 2 mg/L， citric acid 1 mg/L， and phytagel 2.6 g/L， pH 5.8 |
ZM培养基［ ZM medium | N6盐4.0 g/L，蔗糖30 g/L，2，4-D 2.0 mg/L，6-BA 0.2 mg/L，植物凝胶 2.6 g/L，pH 5.8 N6 salts 4.0g/L， sucrose 30 g/L， 2，4-D 2.0 mg/L， 6-BA 0.2 mg/L， and phytagel 2.6 g/L， pH 5.8 |
MRS培养基 MRS medium | MS盐4.4 g/L，酸水解酪蛋白300 mg/L，蔗糖30 g/L，L-谷氨酰胺100 mg/L，L-天冬酰胺100 mg/L，6-BA 1 mg/L，植物凝胶2.6 g/L，pH 5.8 MS salts 4.4 g/L， casein hydrolysate 300 mg/L， sucrose 30 g/L， L-glutamine 100 mg/L， L-asparagine 100 mg/L， 6-BA 1.0 mg/L， and phytagel 2.6 g/L， pH 5.8 |
RIM培养基 RIM medium | MS盐2.2 g/L，蔗糖30 g/L，硫酸镁0.3 g/L，IBA 1 mg/L，植物凝胶2.6 g/L，pH 5.8 MS salts 2.2 g/L， sucrose 30 g/L， MgSO4·5H2O 0.3 g/L， IBA 1.0 mg/L， and phytagel 2.6 g/L， pH 5.8 |
Fig. 1 Callus induction of Chishu 2 on 6 different mediaA‒F: Details of Chishu 2 induced for 15 d on different culture media; G‒L: overall status of Chishu 2 embryogenesis induced for 30 d on various culture media. A and G: OS medium; B and H: TA medium; C and I: SI medium; D and J: SB medium; E and K: SI medium; F and L: ZM medium. Scale = 1 cm
Fig. 2 Embryogenic callus induction rates of different broomcorn millet varieties on the SI mediumM1 to M6 are as follows: Jinshu 5, Jinshu 6, Jishu 3, Longmi 6, Chishu 1, and Chishu 2.The data were analyzed by one-way ANOVA (Turkey). Different lowercase letters indicate significant differences at P<0.05 level. The same below
Fig. 3 Distribution diagram of the embryogenic callus induction rates of 39 broomcorn millet varieties
Fig. 4 Ability of inducing callus in different tissuesA‒C: Millet root growth states on SI medium after 10, 20, and 30 d. D‒F: Millet shoot tips growth states on SI medium after 10, 20, and 30 d. G‒I: Millet mesocotyl growth states on SI medium after 10, 20, and 30 d. Scale = 1 cm
Fig. 5 In vitro regeneration system of broomcorn millet Chishu 2A: Mature seeds. B: Callus after 15 d induction. C: Embryonic callus after 30 d induction. D: Embryonic callus transferred to MRS medium. E: Seedlings on MRS medium. F: New root on RIM medium. Scale = 1 cm
Fig. 6 Effects of different infection conditions on the infection efficiency of broomcorn milletA: Agrobacterium strain. B: Concentration of Agrobacterium liquid. C: Acetosyringone concentration. D: Callus treatment (Untreated, ice bath, heat shock and vacuumize). E: Co-culture time
Fig. 7 PCR detection of T0 transgenic plantsM: Marker DL2000. P: Positive control (pBI121). N: Blank control (ddH2O). CK: Negative control (wild type). 1‒20: T0 transgene plants
筛选方式 Screening method | 总愈伤数 Number of calluses | 抗性愈伤数 Number of survival callus | 抗性苗数 Number of survival seedlings | PCR阳性数 Number of PCR positive plants | PCR阳性率 PCR positive frequency （%） | 转化效率 Transformation frequency （%） |
|---|---|---|---|---|---|---|
| SI + Kan 50 mg/L | 92 | 30 | 24 | 3 | 12.50 | 3.26 |
| SI + Kan 100 mg/L | 100 | 22 | 19 | 5 | 26.30 | 5.00 |
| MRS + Kan 20 mg/L | 110 | 27 | 9 | 6 | 66.70 | 5.50 |
| RIM + Kan 50mg/L | 100 | 28 | 15 | 6 | 40.00 | 6.00 |
Table 3 Genetic transformation results of broomcorn millet
筛选方式 Screening method | 总愈伤数 Number of calluses | 抗性愈伤数 Number of survival callus | 抗性苗数 Number of survival seedlings | PCR阳性数 Number of PCR positive plants | PCR阳性率 PCR positive frequency （%） | 转化效率 Transformation frequency （%） |
|---|---|---|---|---|---|---|
| SI + Kan 50 mg/L | 92 | 30 | 24 | 3 | 12.50 | 3.26 |
| SI + Kan 100 mg/L | 100 | 22 | 19 | 5 | 26.30 | 5.00 |
| MRS + Kan 20 mg/L | 110 | 27 | 9 | 6 | 66.70 | 5.50 |
| RIM + Kan 50mg/L | 100 | 28 | 15 | 6 | 40.00 | 6.00 |
Fig. 8 Protocol of genetic transformation for broomcorn milletA: Embryogenic callus for transformation. B: Callus on co-culture medium. C: GUS transient staining. D: Recovery culture. E: Resistance screening. F: Shoot regeneration from resistant callus. G: Rooting. H: GUS staining of positive seedling. I: Flowchart of broomcorn millet genetic transformation. Scale = 1 cm
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