糜子高频胚胎发生品种遗传转化体系的建立

doi:10.13560/j.cnki.biotech.bull.1985.2025-0297

摘要/Abstract

摘要：

目的以具有高频胚胎发生能力的糜子品种为受体，建立高效的糜子遗传转化体系，为糜子分子育种和基因功能研究提供技术支撑。方法以最适合糜子诱导胚性愈伤组织的SI培养基，从39份糜子品种中筛选具有高频胚胎发生能力的材料，比较成熟胚的茎尖、中胚轴、根的诱导胚性愈伤组织的能力差异；以最优材料为受体，分析比较不同菌株、愈伤处理方式、乙酰丁香酮（AS）浓度、菌液浓度、共培养时间等条件对农杆菌侵染效率的影响，构建基于nptII选择标记的遗传转化体系。结果筛选出适合糜子诱导胚性愈伤组织的SI培养基，并从39份糜子品种中，鉴定出3份高频胚胎发生品种（系），其中赤黍2号胚性愈伤组织诱导率最高（77.7%）。以赤黍2号成熟胚诱导的胚性愈伤组织为受体，优化了农杆菌侵染条件，明确了LBA4404菌株、OD₆₀₀为0.2的菌液浓度、100 μmol/L的乙酰丁香酮浓度、42 ℃热激处理5 min及48 h共培养时间为农杆菌侵染的最佳转化条件。建立了nptII为选择标记基因的遗传转化体系，转化效率达到4.94%。结论筛选出具有高频胚胎发生能力的糜子品种赤黍2号，并建立了农杆菌介导的糜子遗传转化体系。

关键词: 糜子, 赤黍2号, 胚性愈伤组织, 农杆菌, 遗传转化, 胚胎发生, 茎尖, 再生

Abstract:

Objective To establish an Agrobacterium-mediated genetic transformation system for broomcorn millet (Panicum miliaceum L.) using high-frequency embryogenic genotypes as recipients, providing technical support for molecular breeding and gene function research in broomcorn millet. Method This study utilized SI medium, optimal for inducing embryogenic callus in broomcorn millet, to screen 39 varieties for high-frequency embryogenic capacity. The efficacy of inducing embryogenic callus from shoot tips, mesocotyls, and roots was compared. The materials having the highest embryogenic potential was selected as recipients, various factors affecting Agrobacterium infection efficiency, such as bacterial strains, callus pretreatment methods, Acetosyringone (AS) concentration, bacterial suspension density, and co-cultivation time, were investigated to develop a genetic transformation system using the nptII selectable marker gene. Result The SI medium was identified as the most suitable for inducing embryogenic callus in broomcorn millet. From the 39 broomcorn millet varieties tested, 3 high-embryogenic lines were selected, with Chishu 2 showing the highest embryogenic frequency (77.7%). Using embryogenic callus derived from mature embryos of Chishu 2 as the recipient, Agrobacterium infection conditions were optimized. The optimal parameters were the LBA4404 strain, a bacterial cell density of OD₆₀₀ = 0.2, 100 μmol/L acetosyringone, a 42 ℃ heat shock treatment for 5 min, and a 48 h co-cultivation period. A genetic transformation system using the nptII as the selectable marker gene was developed, achieving a transformation efficiency of 4.94%. Conclusion The high-embryogenic broomcorn millet variety Chishu 2 is identified as a suitable recipient, and an Agrobacterium-mediated genetic transformation system is established for broomcorn millet.

Key words: broomcorn millet, Chishu 2, embryogenic callus, Agrobacterium tumefaciens, genetic transformation, embryogenesis, shoot tips, regeneration

张欢欢, 穆晓娅, 周静宜, 吕高培, 肖楠, 李敏, 郝曜山, 吴慎杰. 糜子高频胚胎发生品种遗传转化体系的建立[J]. 生物技术通报, 2025, 41(10): 164-174.

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.

图/表 11

表1 本研究所用39份糜子品种

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

表2 糜子组织培养用到的培养基配方

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^［12］

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^［13］

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^［14］

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^［15］

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^［16］

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^［17］

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

表2 糜子组织培养用到的培养基配方

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^［12］

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^［13］

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^［14］

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^［15］

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^［16］

SI medium

ZM培养基^［17］

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

图1 赤黍2号在6种不同培养基上的愈伤诱导情况A‒F：赤黍2号在不同培养基诱导15 d的细节；G‒L：赤黍2号在不同培养基诱导30 d的胚胎发生整体情况。A和G：OS培养基；B和H：TA培养基；C和I：SI培养基；D和J：SB培养基；E和K：SI培养基；F和L：ZM培养基。比例尺=1 cm

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

图2 不同糜子品种在SI培养基上的胚性愈伤组织诱导率M1‒M6依次为：晋黍5号、晋黍6号、冀黍3号、陇糜6号、赤黍1号、赤黍2号。数据采用单因素方差分析（Turkey法），不同小写字母表示在P<0.05水平差异显著。下同

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

图3 39份个糜子材料的胚性愈伤组织诱导率分布图

Fig. 3 Distribution diagram of the embryogenic callus induction rates of 39 broomcorn millet varieties

图4 不同组织诱导愈伤的能力比较A‒C：糜子的根在SI培养基上培养10、20和30 d后的状态；D‒F：糜子的茎尖在SI培养基上培养10、20和30 d后的状态；G‒I：糜子的中胚轴在SI培养基上培养10、20和30 d后的状态。比例尺=1 cm

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

图5 赤黍2号糜子的体外再生体系A：消毒后的糜子种子；B：诱导15 d的愈伤组织；C：诱导30 d的胚性愈伤组织；D：胚性愈伤组织继代至MRS培养基；E：MRS培养基上长出的幼苗；F：RIM培养基上长出的新根。比例尺=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

图6 不同侵染条件对糜子侵染效率的影响A：农杆菌菌株；B：农杆菌菌液浓度；C：乙酰丁香酮浓度；D：愈伤处理方式（包括未处理、冰浴、热激、真空）；E：共培养时间

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

图7 T0代转基因植株的PCR检测M：Marker DL2000；P：阳性对照（pBI121）；N：空白对照（ddH2O）；CK：阴性对照（野生型）；1‒20：T0代转基因植株

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

表3 糜子遗传转化结果统计

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

图8 糜子遗传转化体系A：转化用的胚性愈伤组织；B：共培养阶段的愈伤组织；C：GUS基因瞬时染色；D：恢复培养；E：抗性筛选；F：抗性愈伤出芽；G：生根；H：阳性苗的GUS染色；I：糜子遗传转化流程图。比例尺=1 cm

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

参考文献 35

[1]	王宇卓, 林元香, 薛亚鹏, 等. 山西糜子核心种质分子身份证构建 [J]. 植物学报, 2023, 58(1): 159-168.
	Wang YZ, Lin YX, Xue YP, et al. Construction of molecular ID card of core germplasm of hog millet (Panicum miliaceum) in Shanxi [J]. Chin Bull Bot, 2023, 58(1): 159-168.
[2]	杨清华, 王洪露, 冯佰利. 糜子品质研究进展与展望 [J]. 植物学报, 2023, 58(1): 22-33.
	Yang QH, Wang HL, Feng BL. Progress and prospect of research on the quality of broomcorn millet [J]. Chin Bull Bot, 2023, 58(1): 22-33.
[3]	杨天育. 糜子分子遗传研究进展与展望 [J]. 寒旱农业科学, 2022(10): 32-36.
	Yang TY. Research progress and prospect on molecular genetics of millet (Panicum miliaceum L.) [J]. J Cold Arid Agric Sci, 2022(10): 32-36.
[4]	薛亚鹏, 丁艺冰, 王宇卓, 等. 基于荧光SSR构建中国糜子核心种质DNA分子身份证 [J]. 中国农业科学, 2023, 56(12): 2249-2277.
	Xue YP, Ding YB, Wang YZ, et al. Construction of DNA molecular identity card of core germplasm of broomcorn millet in China based on fluorescence SSR [J]. Sci Agric Sin, 2023, 56(12): 2249-2277.
[5]	Shi JP, Ma XX, Zhang JH, et al. Chromosome conformation capture resolved near complete genome assembly of broomcorn millet [J]. Nat Commun, 2019, 10(1): 464.
[6]	Zou CS, Li LT, Miki D, et al. The genome of broomcorn millet [J]. Nat Commun, 2019, 10(1): 436.
[7]	Chen JF, Liu Y, Liu MX, et al. Pangenome analysis reveals genomic variations associated with domestication traits in broomcorn millet [J]. Nat Genet, 2023, 55(12): 2243-2254.
[8]	Bai YH, Liu SN, Bai Y, et al. Application of CRISPR/Cas12i.3 for targeted mutagenesis in broomcorn millet (Panicum miliaceum L.) [J]. J Integr Plant Biol, 2024, 66(8): 1544-1547.
[9]	Liu Y, Cheng ZX, Chen WY, et al. Establishment of genome-editing system and assembly of a near-complete genome in broomcorn millet [J]. J Integr Plant Biol, 2024, 66(8): 1688-1702.
[10]	王春芳, 刘晶晶, 李伟, 等. 糜子愈伤诱导及再分化最适条件研究 [J]. 中国农学通报, 2020, 36(9): 94-99.
	Wang CF, Liu JJ, Li W, et al. The optimal condition for callus induction and redifferentiation of Panicum miliaceum L [J]. Chin Agric Sci Bull, 2020, 36(9): 94-99.
[11]	吴会琴. 糜子不同外植体再生体系建立及优化 [D]. 杨凌: 西北农林科技大学, 2020.
	Wu HQ. Establishment and optimization of regeneration system of different explants of broomcorn millet [D]. Yangling: Northwest A & F University, 2020.
[12]	Wu J, Chang X, Li C, et al. QTLs related to rice callus regeneration ability: localization and effect verification of qPRR3 [J]. Cells, 2022, 11(24): 4125.
[13]	石磊. 小麦WOX基因挖掘及其对遗传转化效率的影响和作用机制研究 [D]. 北京: 中国农业科学院, 2021.
	Shi L. Identification of WOX genes and their functions and mechanisms in improving genetic transformation efficiency in Triticum aestivum L. [D]. Beijing: Chinese Academy of Agricultural Sciences, 2021.
[14]	Nguyen DQ, Van Eck J, Eamens AL, et al. Robust and reproducible Agrobacterium-mediated transformation system of the C₄ genetic model species Setaria viridis [J]. Front Plant Sci, 2020, 11: 281.
[15]	Silva TN, Kelly ME, Vermerris W. Use of Sorghum bicolor leaf whorl explants to expedite regeneration and increase transformation throughput [J]. Plant Cell Tissue Organ Cult, 2020, 141(2): 243-255.
[16]	Santos CM, Romeiro D, Silva JP, et al. An improved protocol for efficient transformation and regeneration of Setaria italica [J]. Plant Cell Rep, 2020, 39(4): 501-510.
[17]	Du DX, Jin RC, Guo JJ, et al. Infection of embryonic callus with Agrobacterium enables high-speed transformation of maize [J]. Int J Mol Sci, 2019, 20(2): 279.
[18]	贺榆婷, 卫云丰, 张洁, 等. 谷子高效离体再生基因型和培养基的筛选 [J]. 核农学报, 2019, 33(7): 1265-1272.
	He YT, Wei YF, Zhang J, et al. Screening of high efficient genotypes and medium for in vitro regeneration in foxtail millet [J]. J Nucl Agric Sci, 2019, 33(7): 1265-1272.
[19]	Lowe K, Wu E, Wang N, et al. Morphogenic regulators Baby boom and Wuschel improve monocot transformation [J]. Plant Cell, 2016, 28(9): 1998-2015.
[20]	Li JP, Pan WB, Zhang S, et al. A rapid and highly efficient sorghum transformation strategy using GRF4-GIF1/ternary vector system [J]. Plant J, 2024, 117(5): 1604-1613.
[21]	李素娟, 樊秀霞, 王华, 等. 水稻不同品种组培再生和转基因频率研究 [J]. 核农学报, 2013, 27(12): 1817-1827.
	Li SJ, Fan XX, Wang H, et al. Investigation of regeneration and transformation frequencies generated from different genotypes in rice (Oryza sativa L.) [J]. J Nucl Agric Sci, 2013, 27(12): 1817-1827.
[22]	许洁婷, 刘相国, 金敏亮, 等. 不依赖基因型的高效玉米遗传转化体系的建立 [J]. 作物学报, 2022, 48(12): 2987-2993.
	Xu JT, Liu XG, Jin ML, et al. Establishment of genotype-independent high-efficiency transformation system in maize [J]. Acta Agron Sin, 2022, 48(12): 2987-2993.
[23]	Wang K, Shi L, Liang XN, et al. The gene TaWOX5 overcomes genotype dependency in wheat genetic transformation [J]. Nat Plants, 2022, 8(2): 110-117.
[24]	Bull T, Debernardi J, Reeves M, et al. GRF-GIF chimeric proteins enhance in vitro regeneration and Agrobacterium-mediated transformation efficiencies of lettuce (Lactuca spp.) [J]. Plant Cell Rep, 2023, 42(3): 629-643.
[25]	Yang WT, Zhai HW, Wu FM, et al. Peptide REF1 is a local wound signal promoting plant regeneration [J]. Cell, 2024, 187(12): 3024-3038.e14.
[26]	Purnhauser L, Gyulai G. Effect of copper on shoot and root regeneration in wheat, Triticale, rape and tobacco tissue cultures [J]. Plant Cell Tissue Organ Cult, 1993, 35(2): 131-139.
[27]	Kumar Sahrawat A, Chand S. Stimulatory effect of copper on plant regeneration in indica rice (Oryza sativa L.) [J]. J Plant Physiol, 1999, 154(4): 517-522.
[28]	Cho M, Banh J, Yu M, et al. Improvement of Agrobacterium-mediated transformation frequency in multiple modern elite commercial maize (Zea mays L.) inbreds by media modifications [J]. Plant Cell Tissue Organ Cult, 2015, 121(3): 519-529.
[29]	赵成章. 锌元素对籼稻成熟胚愈伤组织绿苗分化率的影响 [J]. 作物学报, 2001, 27(3): 404-407.
	Zhao CZ. Effect of ZnSO₄ on redifferentiation frequency of callus from mature embryo of indica rice [J]. Acta Agron Sin, 2001, 27(3): 404-407.
[30]	Kothari SL, Agarwal K, Kumar S. Inorganic nutrient manipulation for highly improved in vitro plant regeneration in finger millet—Eleusine coracana (L.) Gaertn [J]. Vitro Cell Dev Biol Plant, 2004, 40(5): 515-519.
[31]	殷榕, 颜如玉, 赵丫杰, 等. 玉米成熟胚高效苗再生体系的建立 [J]. 山东农业大学学报: 自然科学版, 2023, 54(1): 25-31.
	Yin R, Yan RY, Zhao YJ, et al. Establishment of high-efficiency shoots regeneration system from mature embryo of maize (Zea mays L.) [J]. J Shandong Agric Univ Nat Sci Ed, 2023, 54(1): 25-31.
[32]	张东武, 刘辉, 赵惠贤. 小麦成熟胚组织培养再生体系的优化及高再生率基因型的筛选 [J]. 麦类作物学报, 2011, 31(5): 847-852.
	Zhang DW, Liu H, Zhao HX. Optimization of regeneration system of tissue culture from mature embryos and screening of wheat genotypes with high regeneration frequency [J]. J Triticeae Crops, 2011, 31(5): 847-852.
[33]	Zhang WJ, Dewey RE, Boss W, et al. Enhanced Agrobacterium-mediated transformation efficiencies in monocot cells is associated with attenuated defense responses [J]. Plant Mol Biol, 2013, 81(3): 273-286.
[34]	陈倩楠, 王轲, 汤沙, 等. 以抗除草剂Bar基因稳定转化谷子技术研究 [J]. 作物学报, 2018, 44(10): 1423-1432.
	Chen QN, Wang K, Tang S, et al. Use of bar gene for the stable transformation of herbicide-resistant foxtail millet plants [J]. Acta Agron Sin, 2018, 44(10): 1423-1432.
[35]	杨雅文, 朱东杰, 潘弘, 等. 玉米无基因型限制遗传转化体系建立和应用 [J]. 作物学报, 2024, 50(11): 2674-2683.
	Yang YW, Zhu DJ, Pan H, et al. Genotype-independent transformation technique development and application in maize [J]. Acta Agron Sin, 2024, 50(11): 2674-2683.