Scientia Agricultura Sinica ›› 2020, Vol. 53 ›› Issue (18): 3638-3649.doi: 10.3864/j.issn.0578-1752.2020.18.002

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

Generation of Marker-Free Transgenic Barley Plants by Agrobacterium-Mediated Transformation

GONG Qiang1,2(),WANG Ke2(),YE XingGuo2,DU LiPu2,XU YanHao1()   

  1. 1College of Agriculture, Yangtze University, Jingzhou 434025, Hubei
    2Institute of Crop Sciences, Chinese Academy of Agricultural Sciences, Beijing 100081
  • Received:2019-11-15 Accepted:2020-03-09 Online:2020-09-16 Published:2020-09-25
  • Contact: Ke WANG,YanHao XU E-mail:gongqiang0206@163.com;wangke03@caas.cn;xyh09@yangtzeu.edu.cn

RichHTML

29

PDF

818

Abstract:

【Objective】Genotype dependence is a very serious problem in barley genetic transformation in which Golden Promise has been mainly used. In addition, the transformation efficiency of barley is still very low in China. Moreover, the generation of marker-free transgenic barley plants is very important to the future commercialization of genetically modified barley due to the considerably increased public concerns. It is necessary to establish an efficient genetic transformation system for generating marker-free transgenic barley plants, expand available barley genotypes for genetic transformation, and provide technical support for functional genomics study and breeding and potential commercialization of genetically modified varieties of barley. 【Method】The composition and auxin adding ratio in the medium used for barley transformation as well as culture regime was optimized to establish an efficient Agrobacterium-mediated genetic transformation system for barley using the immature embryos approximately 14 days post anthesis of a good agronomic trait variety Vlamingh. A double T-DNA vector pWMB123 containing the Bar gene and the GUS gene cassette was introduced into barley by Agrobacterium-mediated transformation to obtain marker-free transgenic barley plants in T1 generation. 【Result】 It is indicated that the shoot induction medium supplemented with 1.0 mg·L-1 KT, 0.5 mg·L-1 6-BA and 0.05 mg·L-1 NAA significantly promoted the differentiation ability of barley transformed calli. The adding of 2.5 mg·L-1 copper sulfate in shoot induction medium lightened the albinism of transgenic barley plantlets. Through the exploration of different hormone ratios and medium components, it was found that the SM1 supplemented with 1.0 mg·L-1 IBA without other auxin had the best rooting effect. A total of 138 immature embryos were transformed with Agrobacterium containing pWMB123 vector, and fourteen transgenic barley plants were obtained with a transformation efficiency of 10.14%. Detection by PCR, Quick Stix strips and histochemical staining assays confirmed that all T0 plants contained Bar gene, among which four plants did not contain GUS gene. The co-transformation efficiency of the two T-DNAs is 71.43%. In the T1 populations derived from the four T0 transgenic lines containing Bar and GUS genes, two transgenic plants with GUS gene and without Bar gene were screened out in line BL8, and the efficiency for marker-free transgenic plants was 6.9%. The two foreign genes of Bar and GUS were identified to be integrated in most T1 transgenic barley plants be multiple copies using Southern blot. It was further confirmed that BL8-15 and BL8-19 were marker-free transgenic plants. 【Conclusion】An efficient genetic transformation system mediated by Agrobacterium-mediated for generating marker-free transgenic plants with healthy roots and less albinism were successfully established in barley.

Key words: barley, transformation, marker-free transgenic, agrobacterium-mediated

Fig. 1

The structure of pWMB123 vector with double T-DNA regions"

Table 1

Composition of the media used for Agrobacterium-mediated barley immature embryo transformation (g·L-1)"

组分
Composition		 侵染液
IM		 共培养培养基
CM		 第一次筛选培养基
SM1		 第二次筛选培养基
SM2		 过渡培养基
TM		 分化培养基
DM
MS 0.434 0.434 4.340 4.340 2.700 2.170
麦芽糖 Maltose 30 30 20 20
葡萄糖 Glucose 10 10
乙酰丁香酮Acetosyringone 0.02745 0.03924
谷氨酰胺 Glutamine 0.75
酪蛋白水解物Casein hydrolysate 1 1
肌醇 Myoinositol 0.35 0.35
脯氨酸 Proline 0.69 0.69
盐酸硫胺Thiamine HCl 0.001 0.001 0.004
麦草畏Dicamba 0.0025 0.0025
二氯苯氧乙酸2, 4-D 0.0025
硫酸铜CuSO4 0.00125 0.00125 0.00125 0.00125
6-苄氨基嘌呤6-BA 0.0001
琼脂糖Agarose 8
植物凝胶Phytagel 3.5 3.5 3.5 3.0
草铵膦Phosphinothricina 0.005 0.010 0.010 0.005
羧苄青霉素Carbenicillina 0.25 0.25 0.25 0.25
头孢噻肟Cefotaximea 0.25

Table 2

Composition of 15 rooting media with different hormone ratios on the basis of 1/2 MS medium (mg·L-1)"

培养基编号 Medium ID IAA IBA NAA
SR1 1 1 1
SR2 1 1 2
SR3 1 2 1
SR4 2 1 1
SR5 1 1 0
SR6 1 0 1
SR7 0 1 1
SR8 1 0 0
SR9 2 0 0
SR10 0 0 1
SR11 0 0 1.5
SR12 0 0 2
SR13 0 1 0
SR14 0 1.5 0
SR15 0 2 0

Fig. 2

Differentiation performance of barley callus on different shoot induction medium A: Differentiation on DMM without the culture step on TM; B: Differentiation on DM without the culture step on TM; C: Differentiation on DMM after the culture step on TM"

Fig. 3

The effect of copper sulfate on the albinism of transgenic barley plants A: 1/2MS medium without copper sulfate; B: 1/2MS medium with 2.5 mg·L-1 copper sulfate"

Fig. 4

Rooting status of transgenic barley plantlets on 15 media with different hormone ratios"

Fig. 5

Effect of different media containing various auxin ratios on the rooting of transgenic barley plantlets A: DM with IBA; B: BRM with IBA; C: SM1 with IBA and without other auxins"

Fig. 6

Identification of T0 transgenic barley plants A: Detection of Bar protein by Quick Stix strips; B: Detection of GUS gene by histochemical staining; C: Detection of GUS gene by PCR. 1-14: T0 transgenic barley plants; WT: Vlamingh; P: pWMB123 plasmid; M: DL5000 marker"

Fig. 7

PCR detection of Bar (A) and GUS (B) genes in T1 transgenic barley plants 1-22: BL-8 of T1 transgenic barley plants; 23: Vlamingh; P: pWMB123 plasmid; M: DL2000 marker; 15 and 19 were marker-free transgenic plants "

Table 3

Genotyping detection for the Bar and GUS genes in the selected T1 lines by PCR"

株系 Lines Bar+GUS+ Bar+GUS— Bar—GUS+ Bar—GUS—
BL1 19 1 0 0
BL6 23 0 0 0
BL-7 24 2 0 0
BL-8 27 0 2 0

Fig. 8

Southern blot detection of T1 transgenic barley plants A: Southern blot of Bar; B: Southern blot of GUS. WT: Wild type; M: Marker; BL8-15 and BL8-19were marker-free transgenic plants"

[1] 国家大麦青稞产业技术体系. 中国现代农业产业可持续发展战略研究·大麦青稞分册, 北京: 中国农业出版社, 2018.
China Agriculture Research System Of Barley. China's Modern Agricultural Industry Sustainable Development Strategy Research · Barley Volume. Beijing: China Agriculture Press, 2018: 2-8. (in Chinese)
[2] MASCHER M, GUNDLACH H, HIMMELBACH A, BEIER S, TWARDZIOK S O, WICKER T, RADCHUK V, DOCKTER C, HEDLEY P E, RUSSELL J, BAYER M, RAMSAY L, LIU H, HABERER G, ZHANG X Q, ZHANG Q, BARRERO R A, LI L, TAUDIEN S, GROTH M, FELDER M, HASTIE A, SIMKOVA H, STANKOVA H, VRANA J, CHAN S, MUNOZ-AMATRIAIN M, OUNIT R, WANAMAKER S, BOLSER D, COLMSEE C, SCHMUTZER T, ALIYEVA-SCHNORR L, GRASSO S, TANSKANEN J, CHAILYAN A, SAMPATH D, HEAVENS D, CLISSOLD L, CAO S, CHAPMAN B, DAI F, HAN Y, LI H, LI X, LIN C, MCCOOKE J K, TAN C, WANG P, WANG S, YIN S, ZHOU G, POLAND J A, BELLGARD M I, BORISJUK L, HOUBEN A, DOLEZEL J, AYLING S, LONARDI S, KERSEY P, LANGRIDGE P, MUEHLBAUER G J, CLARK M D, CACCAMO M, SCHULMAN A H, MAYER KF X, PLATZER M, CLOSE T J, SCHOLZ U, HANSSON M, ZHANG G, BRAUMANN I, SPANNAGL M, LI C, WAUGH R, STEIN N. A chromosome conformation capture ordered sequence of the barley genome. Nature, 2017,544(7651):427-433.
doi: 10.1038/nature22043 pmid: 28447635
[3] WAN Y, LEMAUX P G. Generation of large numbers of independently transformed fertile barley plants. Plant Physiology, 1994,104(1):37-48.
doi: 10.1104/pp.104.1.37 pmid: 12232059
[4] TINGAY S, MCELROY D, KALLA R, FIEG S, WANG M, THORNTON S, BRETTELL R. Agrobacterium tumefaciens-mediated barley transformation. The Plant Journal, 1997,11(6):1369-1376.
doi: 10.1046/j.1365-313X.1997.11061369.x
[5] TRIFONOVA A, MADSEN S, OLESEN A. Agrobacterium -mediated transgene delivery and integration into barley under a range of in vitro culture conditions. Plant Science, 2001,161(5):871-880.
doi: 10.1016/S0168-9452(01)00479-4
[6] FANG Y D, AKULA C, ALTPETER F. Agrobacterium -mediated barley (Hordeum vulgare L.) transformation using green fluorescent protein as a visual marker and sequence analysis of the T-DNA: barley genomic DNA junctions. Journal of Plant Physiology, 2002,159(10):1131-1138.
doi: 10.1078/0176-1617-00707
[7] TRAVELLA S, ROSS S M, HARDEN J, EVERETT C, SNAPE J W, HARWOOD W A. A comparison of transgenic barley lines produced by particle bombardment and Agrobacterium-mediated techniques. Plant Cell Reports, 2005,23(12):780-789.
doi: 10.1007/s00299-004-0892-x
[8] BARTLETT J G, ALVES S C, SMEDLEY M, SNAPE J W, HARWOOD W A. High-throughput Agrobacterium -mediated barley transformation. Plant Methods, 2008,4(1):22.
doi: 10.1186/1746-4811-4-22
[9] HENSEL G, VALKOV V, MIDDLEFELL-WILLIAMS J, KUMLEHN J. Efficient generation of transgenic barley: The way forward to modulate plant-microbe interactions. Journal of Plant Physiology, 2008,165(1):71-82.
doi: 10.1016/j.jplph.2007.06.015 pmid: 17905476
[10] 马春业. 农杆菌介导miR396基因对大麦愈伤组织的遗传转化[D]. 武汉: 华中农业大学, 2016.
MA C Y. Genetic transformation of miR396 gene into barley callus by Agrobacterium tumefaciens infection[D]. Wuhan: Huazhong Agricultural University, 2016. (in Chinese)
[11] 马玲珑, 任盼荣, 汪军成, 孟亚雄, 马小乐, 李葆春, 王化俊. 啤酒大麦品种甘啤4号成熟胚再生体系的建立. 分子植物育种, 2015,13(3):663-669.
MA L L, REN P R, WANG J C, MENG Y X, MA X L, LI B C, WANG H J. Establishing regeneration system from mature embryos of beer barley variety Ganpi 4. Molecular Plant Breeding, 2015, 2015,13(3):663-669. (in Chinese)
[12] 余桂红, 张旭, 孙晓波, 马鸿翔. 大麦苏啤4号幼胚愈伤组织的诱导及植株的高频再生. 江苏农业学报, 2013,29(5):953-956.
YU G H, ZHANG X, ZHANG X B, MA H X. Calli induction from immature embryos and plantlet high frequency regeneration of Hordeum vulgare L. cv.Supi 4. Jiangsu Journal of Agricultural Science, 2013,29(5):953-956. (in Chinese)
[13] 王兴珍, 林国梁, 赖勇, 杨轲, 孟亚雄, 李葆春, 马小乐, 尚勋武, 王化俊. 西北地区特色大麦品种成熟胚离体培养的初步研究. 麦类作物学报, 2013,33(2):286-289.
doi: 10.7606/j.issn.1009-1041.2013.02.013
WANG X Z, LIN G L, LAI Y, YANG K, MENG Y X, LI B C, MA X L, SHANG X W, WANG H J. Preliminary study of tissue culture for mature embryo from local feature barley. Journal of Triticeae Crops, 2013,33(2):286-289. (in Chinese)
doi: 10.7606/j.issn.1009-1041.2013.02.013
[14] 黎冬华, 廖玉才, 李和平. 根癌农杆菌介导的大麦茎尖转化研究. 麦类作物学报, 2012,32(1):44-47.
doi: 10.7606/j.issn.1009-1041.2012.01.008
LI D H, LIAO Y C, LI H P. Transformation of shoot apical meristems of elit barley cultivars via agribacterium-mediated transformation. Journal of Triticeae Crops, 2012,32(1):44-47. (in Chinese)
doi: 10.7606/j.issn.1009-1041.2012.01.008
[15] 高润红, 杜志钊, 郭桂梅, 邹磊, 何婷, 陈志伟, 李梁, 陆瑞菊, 黄剑华. 优良大麦品种花30幼胚遗传转化体系的优化. 植物遗传资源学报, 2012,13(5):901-906.
GAO R H, DU Z Z, GUO G M, ZOU L, HE T, CHEN Z W, LI L, LU R J, HUANG J H. Optimization of genetic transformation system for immature embryos of improved barley( Hordeum vulgare L.) variety Hua 30. Journal of Plant Genetic Resources, 2012,13(5):901-906. (in Chinese)
[16] 闫永荣, 张正英, 李静雯, 李淑洁. 甘啤4号大麦幼胚愈伤组织诱导及植株再生的研究. 分子植物育种, 2010,8(1):89-93.
YAN Y R, ZHANG Z Y, LI J W, LI S J. Studies on callus induction and plant regeneration from immature embryos of Ganpi4 hao. Molecular Plant Breeding, 2010,8(1):89-93. (in Chinese)
[17] 李静雯, 张正英. 根癌农杆菌介导的大麦幼胚遗传转化影响因素研究. 大麦与谷类科学, 2010(2):1-6.
LI J W, ZHANG Z Y. Study on factors influencing the genetic transformation of immature barley embryos mediated byAgrobacterium tumefaciens. Barley and Cereal Science, 2010(2):1-6. (in Chinese)
[18] 郭晓琳, 张红伟, 刘欣洁, 刘亚娟, 张锋, 孙东发, 谭振波. 大麦成熟胚愈伤组织的诱导和植株再生的研究. 植物遗传资源学报, 2005,6(4):418-422.
GUO X L, ZHANG H W, LIU X J, LIU Y J, ZHANG F, SUN D F, TAN Z B. Study on callus induction and plant regeneration from mature embryos of barley elite cultivars. Journal of Plant Genetic Resources, 2005,6(4):418-422. (in Chinese)
[19] 李会勇, 尹钧, 刘雷, 任江萍, 郭向云, 李志远. Cu2+浓度对啤酒大麦幼胚组织培养与植株再生的影响 . 麦类作物学报, 2003,23(2):27-29.
doi: 10.7606/j.issn.1009-1041.2003.02.047
LI H Y, YIN J, LIU L, REN J P, GUO X Y, LI Z Y. Effect of Cu2+concentration on tissue culture and plant regeneration of beer barley . Journal of Triticeae Crops, 2003,23(2):27-29. (in Chinese)
doi: 10.7606/j.issn.1009-1041.2003.02.047
[20] 严华军, 王君晖. 大麦成熟胚胚性愈伤组织的高频诱导和植株再生. 作物学报, 1996,22(1):58-65.
YAN H J, WANG J H. Efficeient induction of embryogenic callus and regeneration from barley mature embryos. Acta Agronomoca Sinica, 1996,22(1):58-65. (in Chinese)
[21] REN J, LIU L, YIN J, WANG X, LI L. Optimization of transgenic system of barley and transformation of TrxS gene by means of particle bombardment. Acta Botanica Boreali-Occidentalia Sinica, 2004,24(9):1662-1668.
[22] 孔维威. TrxS的表达及其对啤酒大麦发芽种子水解酶和贮藏物质降解的影响[D]. 郑州: 河南农业大学, 2006.
KONG W W. The expression and the effects of TrxS gene on hydrolytic enzyme and degration of storage substances during malting barley seed germination[D]. Zhengzhou: Henan Agricultural University, 2006. (in Chinese)
[23] 吕维涛, 刘芳, 崔德才, 赵檀方. 农杆菌介导法获得转反义磷脂酶Dγ基因大麦. 生物技术, 2005,15(5):11-13.
LÜ W T, LIU F, CUI D C, LIU T F. Production of transgenic barley with Anti -PLDγ gene via Agrobacterium tumefaciens. Biotechnology, 2005,15(5):11-13. (in Chinese)
[24] 李静雯, 张正英, 令利军, 李淑洁. 利用RNAi抑制B-hordein合成降低大麦籽粒蛋白质含量. 中国农业科学, 2014,47(19):3746-3756.
doi: 10.3864/j.issn.0578-1752.2014.19.003
LI J W, ZHANG Z Y, LING L J, LI S J. Creating low grain protein content barley by suppressing b-hordein synthesis through RNA interference. Scientia Agricultura Sinica, 2014,47(19):3746-3756. (in Chinese)
doi: 10.3864/j.issn.0578-1752.2014.19.003
[25] 叶兴国, 陈明, 杜丽璞, 徐惠君. 小麦转基因方法及其评述. 遗传, 2011,33(5):422-430.
YE X G, CHEN M, DU L P, XU H J. Description and evaluation of transformation approaches used in wheat. Hereditas, 2011,33(5):422-430. (in Chinese)
[26] WANG K, LIU H, DU L, YE X G. Generation of marker-free transgenic hexaploid wheat via an Agrobacterium-mediated co- transformation strategy in commercial Chinese wheat varieties. Plant Biotechnology Journal, 2016,15(5):614-623.
doi: 10.1111/pbi.12660 pmid: 27862820
[27] MCCORMAC A C, FOWLER M R, CHEN D F, ELLIOTT M C. Efficient Co-transformation of Nicotiana tabacum by two independent T-DNAs, the effect of T-DNA size and implications for genetic separation. Transgenic Research, 2001,10(2):143-155.
doi: 10.1023/A:1008909203852
[28] XING A, ZHANG Z, SATO S, STASWICK P, CLEMENTE T. The use of the two T-DNA binary system to derive marker-free transgenic soybeans. Vitro Cellular & Developmental Biology Plant, 2000,36(6):456-463.
[29] MILLER M, TAGLIANI L, WANG N, BERKA B, BIDNEY D, ZHAO Z Y. High efficiency transgene segregation in Co-transformed maize plants using an Agrobacterium tumefaciens 2 T-DNA binary system. Transgenic Research, 2002,11(4):381-396.
doi: 10.1023/A:1016390621482
[30] LU L, WU X, YIN X, MORRAND J, CHEN X, FOLK W R, ZHANG Z J. evelopment of marker-free transgenic sorghum [Sorghum bicolor (L.) Moench] using standard binary vectors with bar as a selectable marker. Plant Cell Tissue & Organ Culture, 2009,99(1):97-108.
[31] MANGU V R R, CHIDAMBARAM P, RAJASEKARAN S, KARUPPANNAN V. Transgene stacking and marker elimination in transgenic rice by sequential Agrobacterium-mediated co-transformation with the same selectable marker gene. Plant Cell Reports, 2011,30(7):1241-1252.
doi: 10.1007/s00299-011-1033-y
[32] ISHIDA Y, TSUNASHIMA M, HIEI Y, KOMARI T. Wheat (Triticum aestivum L.). Methods in Molecular Biology, Agrobacterium Protocols, 3rd edn. New York: Springer, 2014: 189-198.
[33] MATTHEWS P R, WANG M B, WATERHOUSE P M, THORNTON S, FIEG S J, GUBLER F, JACOBSEN J V. Marker gene elimination from transgenic barley, using co-transformation with adjacent `twin T-DNAs' on a standard Agrobacterium transformation vector. Molecular Breeding, 2001,7(3):195-202.
doi: 10.1023/A:1011333321893
[34] YAO Q, CONG L, CHANG J L, LI K X, YANG G X, HE G Y. Low copy number gene transfer and stable expression in a commercial wheat cultivar via particle bombardment. Journal of Experimental Botany, 2006,57(14):3737-3746.
doi: 10.1093/jxb/erl145 pmid: 17032730
[35] YAO Q, CONG L, HE G, CHANG J, LI K, YANG G. Optimization of wheat co-transformation procedure with gene cassettes resulted in an improvement in transformation frequency. Molecular Biology Reports, 2007,34(1):61-67.
doi: 10.1007/s11033-006-9016-8
[1] YAN YanGe, ZHANG ShuiQin, XU Meng, XU JiuKai, LI YanTing, ZHAO BingQiang, YUAN Liang. Transformation Characteristics of Dextran-Modified Urea in Fluvo- Aquic Soil [J]. Scientia Agricultura Sinica, 2026, 59(5): 1048-1059.
[2] WANG WeiMeng, WEI YunXiao, TANG YunNi, LIU MiaoMiao, CHEN QuanJia, DENG XiaoJuan, ZHANG Rui. Establishment and Rooting Optimization of Agrobacterium rhizogenes Transformation System in Cotton [J]. Scientia Agricultura Sinica, 2025, 58(8): 1479-1493.
[3] TONG ZhaoYang, LIU WenHua, ZHANG GuoXin, DONG ChunYan, ZHANG YanXia, XU XiaoWei, HE Dong, LIU HeChun, LI Yang, WANG FengTao, FENG Jing, YAO XiaoBo, LIU MeiJin, LIN RuiMing. The Relationship Between Occurrence of Hulless Barley Ear Rot and Population Migration of Grass Mite (Siteroptes spp.) [J]. Scientia Agricultura Sinica, 2025, 58(3): 493-506.
[4] LI MingLi, WEN CaiYun, MA DongHao, LI CunJun, WANG YuWen, KANG Lu, LU Miao. Construction of Salinity Prediction Model Based on Optimal Selection of Soil Hyperspectral Characteristic Bands [J]. Scientia Agricultura Sinica, 2025, 58(20): 4054-4069.
[5] ZOU PeiYi, LIU MeiYan, WANG Ying, LI RanHong. Cloning and Functional Study of AkNAC2 from Actinidia kolomikta [J]. Scientia Agricultura Sinica, 2025, 58(19): 3985-3999.
[6] YANG WenJuan, GAO JiaCheng, WANG YanTing, LI Yan, GUO Ming, WANG JunCheng, MENG YaXiong, WANG HuaJun, SI ErJing. Function of Effector Pg00778 Regulation on the Pathogenicity of Pyrenophora graminea to Barley [J]. Scientia Agricultura Sinica, 2025, 58(15): 3020-3035.
[7] ZHAO YuLei, XIN JiaLu, LI ChengNan, LI Shan, XIE XuFei, YIN Xiao. Screening of Target Genes Downstream of VviERF045, a Transcription Factor Associated with Gray Mold Resistance in Vitis vinifera [J]. Scientia Agricultura Sinica, 2025, 58(13): 2578-2590.
[8] HAN LiJie, CAI HongWei. Progress on Genetic Transformation of Sorghum [J]. Scientia Agricultura Sinica, 2024, 57(3): 454-468.
[9] ZHAO Jie, ZHAO LongYuan, PAN NingHui, GUAN LiRong, DU YunLong, LI ChengYun, WANG YunYue, XIE Yong. Hydrolase Gene BGIOSGA023826 Involved in Regulation of Resistance Process to Rice Blast [J]. Scientia Agricultura Sinica, 2024, 57(23): 4607-4618.
[10] ZHANG AiHong, YANG Fei, ZHAO YuanYe, ZHAO YiHan, DI DianPing, MIAO HongQin. Pathogenicity and Epidemic Risk of Barley Yellow Striate Mosaic Virus [J]. Scientia Agricultura Sinica, 2024, 57(23): 4686-4697.
[11] XU JinQing, BIAN HaiYan, CHEN TongRui, WANG Lei, WANG HanDong, YOU En, DENG Chao, TANG YouLin, SHEN YuHu. Comparison of the Genome Sequence Polymorphisms Between the Main Naked Barley Varieties Kunlun 14 and Kunlun 15 in Qinghai Province [J]. Scientia Agricultura Sinica, 2024, 57(21): 4192-4204.
[12] ZHANG Yi, LIU Ying, CHENG CunGang, LI YanQing, LI Zhuang. Effects of Combined Application Proportion of Cow Manure and Chemical Fertilizer on Soil Organic Carbon Pool and Enzyme Activity in Apple Orchard [J]. Scientia Agricultura Sinica, 2024, 57(20): 4107-4118.
[13] CAO Peng, ZHOU JinHuan, WANG XinLiang, LI ChuXin, LI JiaXIN, JIANG Pei, LIU JinXiang, SONG Zhen. Optimization and Application of Rapid Evaluation System for Citrus Huanglongbing Resistance Mediated by Agrobacterium rhizogenes [J]. Scientia Agricultura Sinica, 2024, 57(16): 3182-3191.
[14] WANG XiaoXuan, ZHANG Min, ZHANG XinYao, WEI Peng, CHAI RuShan, ZHANG ChaoChun, ZHANG LiangLiang, LUO LaiChao, GAO HongJian. Effects of Different Varieties of Phosphate Fertilizer Application on Soil Phosphorus Transformation and Phosphorus Uptake and Utilization of Winter Wheat [J]. Scientia Agricultura Sinica, 2023, 56(6): 1113-1126.
[15] XING MiaoMiao, XU YuanYuan, LU YuYu, YAN JiYong, ZENG AiSong. Development of Ogura CMS Restorers of Broccoli via Genetic Transformation of Rfo [J]. Scientia Agricultura Sinica, 2023, 56(15): 2966-2976.
Viewed
Full text


Abstract

Cited
  Shared   
  Discussed   
No Suggested Reading articles found!
京ICP备09089781号-30
Copyright 2018 © Editorial office of Scientia Agricultura Sinica
Tel: 86-010-82106279    E-mail: zgnykx@mail.caas.net.cn
Supported by Beijing Magtech Co.Ltd