Efficiency of transformation mediated by Agrobacterium tumefaciens using vacuum infiltration in rice (Oryza sativa L.)

Safitri, Fika Ayu;Ubaidillah, Mohammad;Kim, Kyung-Min

  • Received : 2016.01.25
  • Accepted : 2016.02.15
  • Published : 2016.03.31


Agrobacterium-mediated gene transfer has recently been developed to improve rice transformation. In this study, 3 different transformation methods were tested including soaking, co-cultivation, and vacuum infiltration. Agrobacterium tumefaciens GV3101 harboring the binary vector pGreen:: LeGSNOR was used in this experiment. This study aimed to identify the most appropriate method for transferring LeGSNOR into rice. Vacuum infiltration of the embryonic calli for 5 min in Ilpum resulted in high transformation efficiency based on confirmation by PCR, RT-PCR, and qRT-PCR analyses. In conclusion, we described the development of an efficient transformation protocol for the stable integration of foreign genes into rice; furthermore, the study results confirmed that PCR is suitable for efficient detection of the integrated gene. The vacuum infiltration system is a potentially useful tool for future studies focusing on transferring important genes into rice seed calli, and may help reduce time and effort.


Agrobacterium tumefaciens;Transformation;Soaking seeds;Co-cultivation;Vacuum infiltration;Rice


  1. Aldemita RR, Hodges TK (1996) Agrobacterium tumefaciensmediated transformation of japonica and indica rice varieties. Planta 199:612-617
  2. Balaji V, Rjamuni P, Sridevi G, Veluthambi K (2003) Agrobacteriummediated transformation efficiency in blackgram and rice enhanced by multiple copies of pTiBo542 virB and virG. Indian J Biotechnol 2:138-146
  3. Bechtold N, Ellis J, Pelletier G (1993) In-planta Agrobacteriummediated gene transfer by infiltration of adult Arabidopsis thaliana plants. C R Acad Sci Paris Life Sci 316:1194-1199
  4. Bechtold N, Pelletier G (1998) In-planta Agrobacterium-mediated transformation of adult Arabidopsis thaliana plants by vacuum infiltration. Methods Mol Biol 82:259-326
  5. Chen EH, Zhang P, Zuo SM, Li AH, Zhang YF, Chen ZX, Pan XB (2004) Factors affecting Agrobacterium-mediated transformation efficiency in rice. Rice Science 11:181-185
  6. Cheng M, Lowe BA, Spencer M, Ye X, Amstrong CL (2004) Invited review: factors influencing Agrobacterium-mediated transformation of monocotyledonous species. In Vitro Cell Dev Biol Plant 40:31-45
  7. De La Riva GA, Gonzalez-Cabrera J, Vazquez-Padron R, Ayra-Pardo C (1998) Agrobacterium tumefaciens: a natural tool for plant transformation. Electron J Biotechin 1:24-25
  8. Dean JD, Goodwin PH, Hsiang T (2002) Comparison of relative RT-PCR and northern blot analyses to measure expression of ${\beta}$-1,3-glucanase in Nicotiana benthamiana infected with Colltotrichum destructivum. Plant Mol Biol Rep 20:347-356
  9. Dey M, Bakshi S, Galiba G, Sahoo L, Panda SK (2012) Development of a genotype independent and transformation amenable regeneration system from shoot apex in rice (Oryza sativa spp. indica) using TDZ. 3 Biotech 2:233-240
  10. Duan Y, Zhai C, Li H, Li J, Mei W, Gui H, Ni D, Song F, Li L, Zhang W, Yang J (2012) An efficient and high-throughput protocol for Agrobacterium-mediated transformation based on phosphomannose isomerase positive selection in japonica rice (Oryza sativa L). Plant Cell Rep 31:1611-1624
  11. Escobar MA, Dandekar AM (2003) Agrobacterium tumefaciens as an agent of diseases. Trends Plant Sci 8:380-386
  12. Fullner KJ, Nester EW (1996) Temperature affects the T-DNA transfer machinery of Agrobacterium tumefaciens. J. Bacteriol. 178:1498-1504
  13. Gong B, Wen D, Wang X, Wei M, Yang F, Li F, Shi Q (2015) S-Nitrosoglutathione Reductase-modulated redox signaling controls sodic alkaline stress responses in Solanum lycopersicum L. Plant Cell Physiol 56:790-802
  14. Hamilton CM, Frary A, Lewis C, Tanksley SD (1996) Stable transfer of intact molecular weight DNA into plant chromosomes. Proc Natl Acad Sci USA 93:9975-9979
  15. Hancock JT (2012) NO synthase? generation of nitric oxide in plants. Period Biol 114:19-24
  16. Hiei Y, Ishida Y, Komari T (2014) Progress of cereal transformation technology mediated by Agrobacterium tumefaciens. Front Plant Sci 5:628
  17. Hiei Y, Ohta S, Komari T, Kumashiro T (1994) Efficient transformation of rice (Oryza sativa L.) mediated by Agrobacterium and sequence analysis of the boundaries of the T-DNA. Plant J 6:271-282
  18. Hopkins WG, Huner NPA (2009) Introduction to plant physiology. John wiley & Sons, Inc., Hoboken, pp 281-283
  19. Hoque ME, Mansfield JW, Bennett MH (2005) Agrobateriummediated transformation of indica rice genotypes: an assessment of factors affecting the transformation efficiency. Plant Cell Tiss Org 82:45-55
  20. Huang JQ, Wei ZM, An HL, Zhu YX (2001) Agrobacterium tumefaciens-mediated transformation of rice with the spider insecticidal gene conferring resistance to leaf folder and striped stem borer. Cell Res 11:149-155
  21. Karthikeyan A, Pandian SK, Ramesh M (2011) Agrobacteriummediated transformation of leaf base derived callus tissues of popular indica rice (Oryza sativa L. sub sp. indica cv. ADT 43). Plant science 181:258-268
  22. Kim YJ, Lee OR, Kim KT, Yang DC (2012) High frequency of plant regeneration through cyclic secondary somatic embryogenesis in Panax ginseng. J Ginseng Res 36:442-448
  23. Kubienova L, Kopecny D, Tylichova M, Briozzo P, Skopalova J, Sebela M, Navratil M, Tache R, Luhova L, Barroso JB, Petrivalsky M (2013) Structural and functional characterization of a plant S-nitrosoglutathione reductase from Solanum lycopersicum. Biochimie 95:889-902
  24. Kubo M, Purevdorj M (2004) The Future of Rice Production and Consumption. Journal of Food Distribution Research 35:128-142
  25. Lin J, Zhou B, Yang Y, Mei J, Zhao X, Guo X, Huang X, Tang D, Liu X (2009) Piercing and vacuum infiltration of the mature embryo: a simplified method for Agrobacterium-mediated transformation of indica rice. Plant Cell Rep 28:1065-1074
  26. Manimaran P, Kumar GR, Reddy MR, Jain S, Rao TB, Mangrauthia SK, Sundaram RM, Ravichandran S, Balachandran SM (2013) Infection of early and young callus tissues of indica rice BPT 5204 enhances regeneration and transformation efficiency. Rice Science 20:415-426
  27. Mariashibu TS, Subramanyam K, Arun M, Mayavan S, Rajesh M, Theboral J, Manickavasagam M, Ganpathi A (2013) Vacuum infiltration enhances the Agrobacterium-mediated genetic transformation in Indian soybean cultivars. Acta Physiol Plant 35:41-54
  28. Nishimura A, Aichi I, Matsuoka M (2007) A Protocol for Agrobacterium-mediated transformation in rice. Nat Protoc 1:2796-2802
  29. Njimona L, Lamparter T (2011) Temperature effects on Agrobacterium Phytochrome Agp1. PloS One 6:25977
  30. Sahoo KK, Tripathi AK, Pareek A, Sopory SK, Pareek SLS (2011) An improved protocol for efficient transformation and regeneration of diverse indica rice cultivars. Plant Methods 7:49
  31. Saika H, Toki S (2010) Mature seed-derived callus of the model indica rice variety Kasalath is highly competent in Agrobacteriummediated transformation. Plant Cell Rep 29:1351-1364
  32. Santos CF, Sakai VT, Machado MAAM, Schippers DN, Greene AS (2004) Reverse transcription and polymerase chain reaction:principles and applications in dentistry. J Appl Oral Sci 12:1-11
  33. Shiao YH (2003) A new reverse transcription-polymerase chain reaction method for accurate quantification. BMC Biotechnology 3.
  34. Shri M, Rai A, Verma PK, Misra P, Dubey S, Kumar S, Verma S, Gautam N, Tripathi RD, Trivedi PK, Chakrabarty D (2013) An improved Agrobacterium-mediated transformation of recalcitrant indica rice (Oryza sativa L.) cultivars. Protoplasma 250:631-636
  35. Tague BW, Mantis J (2006) In-Planta Agrobacterium-mediated transformation by vacuum infiltration. Methods Mol Biol 323:215-223
  36. Tellier R, Bukh J, Emerson SU, Purcell RH (1996) Amplification of the full-length hepatitis A virus genome by long reverse transcription-PCR and transcription of infectious RNA directly from the amplicon. Proc. Natl. Acad. Sci. USA 93:4370-4373
  37. Toki S (1997) Rapid and Efficient Agrobacterium-mediated transformation in rice. Plant Mol Biol Rep 15:16-21
  38. Tzfira T, Li J, Lacxroic B, Citovxky V (2004) Agrobacterium T-DNA integration: molecules and models. Trends Genet 20:375-383

Cited by

  1. Acetosyringone treatment duration affects large T-DNA molecule transfer to rice callus vol.18, pp.1, 2018,