In Planta transformation for conferring salt tolerance to a tissue-culture unresponsive indica rice (Oryza sativa L.) cultivar

  • Tasnim Ahmed
  • Sudip Biswas
  • Sabrina M. Elias
  • M. Sazzadur Rahman
  • Narendra Tuteja
  • Zeba I. Seraj
Plant Tissue Culture


Many farmer-popular indica rice (Oryza sativa L.) cultivars are recalcitrant to Agrobacterium-mediated transformation through tissue culture and regeneration. In planta transformation using Agrobacterium could therefore be a useful alternative for indica rice. A simple and reproducible in planta protocol with higher transformation efficiencies than earlier reports was established for a recalcitrant indica rice genotype. Agrobacterium tumefaciens containing the salt tolerance-enhancing Pea DNA Helicase45 (PDH45) gene, with the reporter and selectable marker genes, gus-INT (β-glucuronidase with intron) and hygromycin phosphotransferase (hpt), respectively, were used. Overnight-soaked mature embryos were infected and allowed to germinate, flower, and set T1 seeds. T0 plants were considered positive for the transgene if the spikelets of one or more of their panicles were positive for gus. Thereafter, selection at T1 was done by germination in hygromycin and transgenic status re-confirmation by subjecting plantlet DNA/RNA to gene-specific PCR, Southern and semi-quantitative RT-PCR. Additionally, physiological screening under saline stress was done at the T2 generation. Transformation efficiency was found to be 30–32% at the T0 generation. Two lines of the in planta transformed seedlings of the recalcitrant rice genotype were shown to be saline tolerant having lower electrolyte leakage, lower Na+/K+, minimal leaf damage, and higher chlorophyll content under stress, compared to the WT at the T2 generation.


Agrobacterium In planta transformation PDH45 Gus Salt stress 





hygromycin phosphotransferase


BRRI Dhan 36


Pea DNA Helicase 45




repeat-induced mutation


high-yielding variety


tissue culture


multiple cloning site


standard evaluation system



Funds for this work are gratefully acknowledged from BAS-USDA (Bangladesh Academy of Sciences and US Department of Agriculture). Thanks also to the Ministry of Science and Technology, Bangladesh, for the award of the National Science and Technology (NST) fellowship 2011-12, to TA. There is no conflict of interest regarding the publication of this manuscript from these funding agencies.

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflicts of interest.

Supplementary material

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Fig. S1

gus positive samples of BR36 T1 generation (a and b) and BA T1 generation (c, d and e). The gus positive transgenic plants were purple (a) blue (b and d) depending on the colored product produced after enzymatic reaction in presence of the substrate X-GLUC. Here two types of X-GLUC substrate were used: ammonium salt of 5-bromo-4-chloro-3-indolyl-beta-D-glucuronic acid and sodium salt of 5-bromo-4-chloro-3-indolyl-beta-D-glucuronic acid. The former one produced purple color while the latter, blue, when cleaved by β-glucuronidase (encoded by gusA). a) BR36 T1 GUS positive spikelet samples (P1-P6) from the panicles of T0 plants showed purple color and WTBR36 showed no color change. b) BR36 T1 GUS positive leaf samples from different hygromycin positive plants (P-3-4, P-6-2, P-6-3 and P-6-5) showed dark blue color whereas wild type BR36 showed no color. c) and d) Germinating transgenic T1 generation Binnatoa from different T0 plants showed blue color (d) whereas wild type Binnatoa showed no color change e) BA T1 GUS positive leaf samples from different hygromycin positive plants (2–1, 2–2, 3–1, 3–2 and 4–1) showed dark blue color whereas wild type BA showed no color. Positive control: GUS transformed tobacco leaf. (GIF 18 kb)

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High Resolution Image (TIFF 433 kb)
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Fig S2

Hygromycin assay of T1 generation plants of BA (a, c and e) and BR36 (b, d, f). Transformants in water control only (a) and (b); transformants (c) and (d) and non-transformants (e) and (f) in hygromycin solution (100 mg/l). Transgenics grew vigorously whereas growth retardation occurred in the wild type plants. (GIF 78 kb)

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High Resolution Image (TIFF 1289 kb)
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Fig. S3

PCR of BA and BR36 T1 and T2 generation plants with hpt specific primer a) BR36 T1 generation PCR positive plants (P2-P6) b) BR36 T2 generation PCR positive plants (different plants germinated from P3, P5 and P6 lines); c) Binnatoa T1 generation PCR positive plants; d) BA T2 generation PCR positive plants. Positive control = pCAMBIA1301-PDH45 vector; WT = wild type BA; Negative control = water control; 1 kb + DNA ladder = marker. (GIF 8 kb)

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High Resolution Image (TIFF 952 kb)
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Fig 4S

Better phenology of transformed T2 BR36 matured plant (P6–1-1): WT BR36 and transgenic P6–1-1 (right). This is one of the replicates of line P6 which had higher plant height, tillers and grains compared to WTBR36 under control conditions (also see Table 4). WT = Wild Type; P6–1-1 = a salt tolerant PDH45-BR36 T2 generation matured plant produced by In planta transformation. (GIF 5 kb)

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High Resolution Image (TIFF 1171 kb)
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Fig S5

PCR from genomic DNA (A) and RT-PCR (B) of BR36 T2 generation plants with PDH45 gene specific primer; P5–1-1, P5–2-1, P-6-1-1, P6–2-1 and P6–3-1.These are T2 progenies of two independently transformed plants, P5 and P6. Positive control = pCAMBIA1301-PDH45 vector; 1 kb+DNA ladder = marker; C) Agrobacterium contamination analysis by polymerase chain reaction with kanamycin resistant gene specific primers in T2 generation transformants; Desired band for kanamycin resistant gene only found in the positive plasmid control, but there is no band found in WT and transgenics BR36 (P5–1-1, P5–2-1, P-6-1-1). (GIF 1 kb)

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High Resolution Image (TIFF 1073 kb)


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Copyright information

© The Society for In Vitro Biology 2017

Authors and Affiliations

  • Tasnim Ahmed
    • 1
  • Sudip Biswas
    • 1
  • Sabrina M. Elias
    • 1
  • M. Sazzadur Rahman
    • 2
  • Narendra Tuteja
    • 3
  • Zeba I. Seraj
    • 1
  1. 1.Plant Biotechnology Laboratory, Department of Biochemistry and Molecular BiologyUniversity of DhakaDhakaBangladesh
  2. 2.Plant Physiology DivisionBangladesh Rice Research InstituteGazipurBangladesh
  3. 3.Amity Institute of Microbial TechnologyAmity UniversityNoidaIndia

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