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Title: Stress-inducible expression of barley Hva1 gene in transgenic mulberry

Supplementary data 2 Table 1 and Fig. S1 to S7. Title: Stress-inducible expression of barley Hva1 gene in transgenic mulberry displays enhanced tolerance against drought, salinity and cold stress Journal name: Transgenic Research Authors:

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Title: Stress-inducible expression of barley Hva1 gene in transgenic mulberry

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  1. Supplementary data 2 Table 1 and Fig. S1 to S7 Title: Stress-inducible expression of barley Hva1 gene in transgenic mulberry displays enhanced tolerance against drought, salinity and cold stress Journal name: Transgenic Research Authors: Vibha G. Checker, Anju K. Chhibbar and Paramjit Khurana Corresponding Authors: Paramjit Khurana Department of Plant Molecular Biology, University of Delhi South Campus, Dhaula Kuan, New Delhi-110021, India.

  2. Supplementary data 2 Table 1: Morphological features of transgenic and non-transgenic lines

  3. DROUGHT STRESS SALT STRESS a a b b c c Supplementary data 2, Fig. S1 Physiological characterization of actin1:Hva1 transgenic lines under drought (2% PEG) and salinity stress (400 mM NaCl). Proline content (a), Membrane injury (b), and Photosynthetic yield (Fv/Fm) (c) were measured at indicated time points. (K2 = Non-transgenic line, ST = actin1:Hva1 transgenic lines) Values are significant at P ≤ 0.05

  4. Right border Left Border (26 bp) Nos-pro (302 bp) Nos-ter (253 bp) Hva 1 (642bp) Nos-ter (253 bp) rd29A-Pro (686bp) NPT II (795bp) (25bp) Sac I BamHI pBI121:rd29a:Hva1 (a) HindIIII (b) (c) (e) (f) (d) (g) (h) ST NT VR9.1 (g) Supplementary data 2, Fig. S2 Transformation and regeneration of mulberry (Morus indica) cv. K-2 via Agrobacterium tumefaciens (pBI121:rd29A:Hva1). (a) Vector map of pBI121:rd29a:Hva1. (b) Non-transformed hypocotyl, cotyledon and calli respectively. (c) Transformed explants after transfer to selection medium. (d) Regenerating explants on shoot elongation medium. (e) Shoot explants on root inducing medium. (f) Regenerants established in earthen pots. (g) Morphology of some transgenic plants. (h) Appearance of non-transgenic (NT), ST (actin1:Hva1) and VR9.1 (rd29A:Hva1) plants

  5. Transgenic plants Negative VR12.1 VR11.3 VR6.2 VR7.1 VR5.5 VR8.1 VR9.1 VR4.2 VR1 VR2 a Positive Ladder 1000 bp Hva 1 500 bp 250 bp Negative Positive Transgenic plants VR11.3 Ladder b VR7.1 VR6.2 VR9.1 VR5.5 VR8.1 VR4 VR1 1000 bp npt II 500 bp Supplementary data 2, Fig. S3 PCR analysis of genomic DNA samples of putative transgenic plants of M. indica cv. K2 using specific primers of Hva1 (a) and nptII (b)

  6. 23.4kb 6.0 4.0 1.5 NT Digested NT Undigested VR 11.3 VR 6.2 VR 5.3 VR 9.1 VR1 VR2 Hva 1 500bp Supplementary data 2, Fig. S4 Molecular confirmation of rd29A:Hva1 overexpressing transgenic mulberry plants (VR) and non-transgenic control plants (NT). Southern hybridization of genomic DNA samples of transgenic plants of mulberry was carried out with Hva1 gene as probe. Lane 1 and 2: NT (Non-transgenic/negative control), lane 3 to 8: BamHI and SacI digested samples of genomic DNA of transformed plantlets

  7. a b c d Supplementary data 2, Fig. S5 Comparison of non-transgenic (NT), actin1:Hva1 (ST) and rd29A:Hva1 (VR) transgenic mulberry plants under control conditions. Proline content (a), percent membrane injury (b), photosynthetic yield (Fv/Fm) (c), and relative water content (RWC) (d) were measured to evaluate metabolic status of plants. Leaf tissues harvested on 0 day of the experiment were used for various analyses. Results are average of three experiments and three independent plants were taken for each experiment. Error bars represent standard deviation. Values are significant at P ≤ 0.05

  8. VR1 VR6.2 NT ST VR7.1 VR8.1 VR9.1 VR11.3 Supplementary data 2, Fig. S6 Western blot analysis to confirm expression of barleyHVA1in transgenic mulberry plants leaf proteinsunder field conditions ST VR9.1 VR1 VR11.3 NT Supplementary data 2, Fig. S7 In situ detection of reactive oxygen species (ROS) by nitroblue tetrazolium (NBT) staining of non-transgenic (NT), actin1:Hva1 (ST) and rd29A:Hva1 (VR) transgenic mulberry leaves. Blue color indicates the characteristic staining pattern of ROS

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