In collaboration with Payame Noor University and Iranian Biotechnology Society

Document Type : Research Paper


1 Assistant Professor, Agricultural Biotechnology Research Institute of Iran (ABRII), Agricultural Research, Education and Extension Organization (AREEO), Karaj, Iran.

2 Associate Professor, National Institute of Genetic Engineering and Biotechnology (NIGEB), Tehran, Iran

3 Associate Professor, Iranian Research Institute of Plant Protection, Agricultural Research, Education and Extension Organization (AREEO), Tehran, Iran.


Sour lime (Citrus aurantifolia L.) is one of the most important woody plants is widely known for its recalcitrance to genetic transformation. We aimed herein to evaluate effective factors influencing the transformation efficiency and the reduction of chimeric transgenic shoots in sour lime. Epicotyl and internode explants were genetically transformed with different Agrobacterium tumefaciens strains e.g., LBA4404, GV3850, and GV3101, harboring the vectors pBI121 and pCAMBIA3301 containing β-glucuronidase (GUS) as a reporter gene. The effect of the following factors was evaluated: Agrobacterium concentration (OD600=0.3, 0.5 and 1), during inoculation (5 seconds, 10 minutes and 30 minutes), co-culture (2 and 3 days), and the selection regime (phosphinothricin at 1, 3, 5 and 10 mg/l and kanamycin at 25, 50, 75 and 100 mg/l). In following, transformation efficiency and the chimeric transgenic shoots rate were respectively confirmed by PCR and GUS assays. The results showed that Agrobacterium strain LBA4404, at the OD600 of 0.5, with 5 seconds (for epicotyl) and 10 minutes (for internode) inoculation at two-day co-culture period, were identified the most suitable treatments for both explants. The transformation frequencies ranged from 0.93% for internode on DKW medium containing 1.0 mg/l of phosphinothricin to 14.29% for epicotyl on DKW medium containing 50 mg/l of kanamycin. Inclusion of the high-level of selective treatments, improved the transformation rate through decreasing frequency escape and chimeric transgenic shoots. These findings provide novel insights into the appropriate procedure to constitute non-chimeric lime transgenic shoots.


Main Subjects

Acanda Y, Canton M, Wu H, Zale J (2017). Kanamycin selection in temporary immersion bioreactors allows visual selection of transgenic citrus shoots. Plant Cell Tiss Org. 129(2): 351-357.
Agricultural Statistics (2019). Ministry of agriculture-jahad of Iran. Volume III. p. 163.
Cevik B, Lee RF, Niblett CL (2012). Agrobacterium-mediated transformation of grapefruit with the wild-type and mutant RNA-dependent RNA polymerase genes of Citrus tristeza virus. Turk J Agric For. 36(2): 195-206.
Cheng CZ, Yang JW, Yan HB, Bei XJ, Zhang YY, Lu ZM, Zhong GY (2015). Expressing p20 hairpin RNA of Citrus tristeza virus confers Citrus aurantium with tolerance/resistance against stem pitting and seedling yellow CTV strains. J. Integr. Agric. 14(9): 1767-1777.
Domínguez A, Cervera M, Pérez R M, Romero J, Fagoaga C, Cubero J, López M M, Juárez J A, Navarro L, Peña L (2004). Characterisation of regenerants obtained under selective conditions after Agrobacterium-mediated transformation of citrus explants reveals production of silenced and chimeric plants at unexpected high frequencies. Mol. Breed. 14(2): 171-183.
Domínguez A, de Mendoza A H, Guerri J, Cambra M, Navarro L, Moreno P, Peña L (2002). Pathogen-derived resistance to Citrus tristeza virus (CTV) in transgenic Mexican lime (Citrus aurantifolia (Christ.) Swing.) plants expressing its p25 coat protein gene. Mol. Breed. 10(1): 1-10.
Dominguez A, Guerri J, Cambra M, Navarro L, Moreno P, Pena L (2000). Efficient production of transgenic citrus plants expressing the coat protein gene of citrus tristeza virus. Plant Cell Rep. 19(4): 427-433.
Doyle JJ, Doyle JL (1990). Isolation of plant DNA from fresh tissue. Focus. 12(13): 39-40.
Dutt M, Ananthakrishnan G, Jaromin M K, Brlansky R H, Grosser J W (2012). Evaluation of four phloem-specific promoters in vegetative tissues of transgenic citrus plants. Tree Physiol. 32(1): 83-93.
Dutt M, Grosser J W (2009). Evaluation of parameters affecting Agrobacterium-mediated transformation of citrus. Plant Cell Tiss Org. 98(3): 331-340.
Dutt M, Vasconcellos M, Grosser JW (2011). Effects of antioxidants on Agrobacterium-mediated transformation and accelerated production of transgenic plants of Mexican lime (Citrus aurantifolia Swingle). Plant Cell Tiss Org. 107(1): 79-89.
Fagoaga C, López C, de Mendoza A H, Moreno P, Navarro L, Flores R, Peña L (2006). Post-transcriptional gene silencing of the p23 silencing suppressor of Citrus tristeza virus confers resistance to the virus in transgenic Mexican lime. Plant Mol. Biol. 60(2): 153-165.
Fang LI, Dai SM, Deng ZN, Li DZ, Long GY, Na LI, Yi LI, Gentile A (2017). Evaluation of parameters affecting Agrobacterium-mediated transient expression in citrus. J. Integr. Agric. 16(3): 572-579.
Ganjeh A, Rahimian H, Basavand E (2021). Agrobacterium tumefaciens causing crown and stem gall disease of citrus propagation nursery in Iran. J. Plant Pathol. 103(1): 355-355.
Ghaderi I, Sohani M M, Mahmoudi A (2018). Efficient genetic transformation of sour orange, Citrus aurantium L. using Agrobacterium tumefaciens containing the coat protein gene of Citrus tristeza virus. Plant Gene 14:7-11.
Ghorbel R, Domínguez A, Navarro L, Peña L (2000). High efficiency genetic transformation of sour orange (Citrus aurantium) and production of transgenic trees containing the coat protein gene of citrus tristeza virus Tree Physiol. 20(17): 1183-1189.
Gutiérrez-e M A, Luth D, Moore G A (1997). Factors affecting Agrobacterium-mediated transformation in Citrus and production of sour orange (Citrus aurantium L.) plants expressing the coat protein gene of citrus tristeza virus. Plant Cell Rep. 16(11): 745-753.
Holsters M, De Waele D, Depicker A, Messens E, Van Montagu M, Schell J, (1978). Transfection and transformation of Agrobacterium tumefaciens. Molec. gen. Genet.163(2): 181-187.
Hu W, Fagundez S, Katin-Grazzini L, Li Y, Li W, Chen Y, Wang X, Deng Z, Xie S, McAvoy RJ, Li Y (2017). Endogenous auxin and its manipulation influence in vitro shoot organogenesis of citrus epicotyl explants. Hortic. Res. 4(1):1-6.
Hu W, Li W, Xie S, Fagundez S, McAvoy R, Deng Z, Li Y (2016). Kn1 gene overexpression drastically improves genetic transformation efficiencies of citrus cultivars. Plant Cell Tiss Org. 125(1): 81-91.
Jardak R, Boubakri H, Zemni H, Gandoura S, Mejri S, Mliki A, Ghorbel A (2020). Establishment of an in vitro regeneration system and genetic transformation of the Tunisian 'Maltese half-blood' (Citrus sinensis): an agro-economically important variety. 3 Biotech. 10(3): 1-11.
Jefferson RA, Kavanagh TA, Bevan MW (1987). GUS fusions: beta-glucuronidase as a sensitive and versatile gene fusion marker in higher plants. EMBO J. 6(13): 3901-3907.
   Lopez C, Cervera M, Fagoaga C, Moreno P, Navarro L, Flores R, Pena L (2010). Accumulation of transgene derived siRNAs is not sufficient for RNAi mediated protection against Citrus tristeza virus in transgenic Mexican lime. Mol. Plant Pathol. 11(1): 33-41.
Niedz RP, Albano JP, Marutani-Hert M (2015). Effect of various factors on shoot regeneration from citrus epicotyl explants. J Appl Hortic 17(2):121-128.
  Oliveira M L P, Moore G, Thomson J G (2015). Agrobacterium-Mediated Transformation of Mexican Lime (Citrus aurantifolia Swingle) Using Optimized Systems for Epicotyls and Cotyledons. Adv Biosci Biotechnol. 6(11): 657.
Onwimol P, Chanprame S, Chanprame S (2017). Agrobacterium-mediated transformation of Cry1Ab gene into Tectona grandis L. (teak). J. Assoc. 23(1): 68-78.
Poles L, Licciardello C, Distefano G, Nicolosi E, Gentile A, La Malfa S (2020). Recent advances of in vitro culture for the application of new breeding techniques in Citrus. Plants. 9(8): 938.
Romero-Romero J L, Inostroza-Blancheteau C, Reyes-Díaz M, Matte J P, Aquea F, Espinoza C, Gil P M, Arce-Johnson P (2020). Increased drought and salinity tolerance in Citrus aurantifolia (Mexican lemon) plants overexpressing arabidopsis CBF3 gene. J. Soil Sci. Plant Nutr. 20(1): 244-252.
Silva R P D, Souza A J D, Mendes B M J, Mourão Filho F D A A (2010). Sour orange bud regeneration and in vitro plant development related to culture medium composition and explant type. Rev Bras Frutic. 32(1): 1-8.
Sohani M M, Rezadoost M H, Zamani A H, Mirzaei M R, Afsharifar A (2015). High efficiency Agrobacterium-mediated transformation of sour orange (Citrus aurantium L.) using gene encoding Citrus Tristeza Virus coat protein. J. Appl. Hortic. 17(2): 109-114.
Wu H, Acanda Y, Canton M, Zale J (2019). Efficient biolistic transformation of immature citrus rootstocks using phosphomannose-isomerase selection. Plants. 8(10): 390.
Zhang Y Y, Zhang D M, Zhong Y, Chang X J, Hu M L, Cheng CZ (2017) A simple and efficient in planta transformation method for pommelo (Citrus maxima) using Agrobacterium tumefaciens. Sci. Hortic. 214: 174-179.