Overexpression of VpPR10.1 by an efficient transformation method enhances downy mildew resistance in V. vinifera
Putrescine and spermidine increase the transformation efficiency of Vitis vinifera L. cv. Thompson seedless. Accumulation of VpPR10.1 in transgenic V. vinifera Thompson seedless, likely increases its resistance to downy mildew.
A more efficient method is described for facilitating Agrobacterium-mediated transformation of Vitis vinifera L. cv. Thompson Seedless somatic embryogenesis using polyamines (PAs). The efficacies of putrescine, spermidine and spermine are identified at a range of concentrations (10 µM, 100 µM and 1 mM) added to the culture medium during somatic embryo growth. Putrescine (PUT) and spermidine (SPD) promote the recovery of proembryonic masses (PEM) and the development of somatic embryos (SE) after co-cultivation. Judging from the importance of the time-frame in genetic transformation, PAs added at the co-cultivation stage have a stronger effect than delayed selection treatments, which are superior to antibiotic treatments in the selection stage. Best embryogenic responses are with 1 mM PUT and 100 µM SPD added to the co-culture medium. Using the above method, a pathogenesis-related gene (VpPR10.1) from Chinese wild Vitis pseudoreticulata was transferred into Thompson Seedless for functional evaluation. The transgenic line, confirmed by western blot analysis, was inoculated with Plasmopara viticola to test for downy mildew resistance. Based on observed restrictions of hyphal growth and increases in H2O2 accumulation in the transgenic plants, the accumulation of VpPR10.1 likely enhanced the transgenic plants resistance to downy mildew.
KeywordsGrapevine Polyamine Transformation Downy mildew VpPR10.1 Resistance
This work was supported by the National Natural Science Foundation of China (Grant Nos. 31471844, 31601716, 31272125). Support was also received from the “948” Program, Ministry of Agriculture, China (Grant No. 2016-X11). The authors thank Dr. Alexander (Sandy) Lang from RESCRIPT Co. (New Zealand) for useful comments and language editing which have greatly improved the manuscript.
Compliance with ethical standards
Conflict of interest
The authors declare that they have no conflict of interest.
- Agüero CB, Meredith CP (2006) Genetic transformation of Vitis vinifera L. cvs Thompson seedless and chardonnay with the pear PGIP and GFP encoding genes. Vitis 45:1–8Google Scholar
- Arun M et al (2016) Involvement of exogenous polyamines enhances regeneration and Agrobacterium. 3 Biotech 6:1–12Google Scholar
- Asselbergh B, Curvers K, França SC, Audenaert K, Vuylsteke M, Van Breusegem F, Höfte M (2007) Resistance to Botrytis cinerea in sitiens, an abscisic acid-deficient tomato mutant, involves timely production of hydrogen peroxide and cell wall modifications in the epidermis. Plant Physiol 144:1863–1877CrossRefPubMedPubMedCentralGoogle Scholar
- Breiteneder H et al (1989) The gene coding for the major birch pollen allergen Betv1, is highly homologous to a pea disease resistance response gene. EMBO J 8:1935Google Scholar
- Chong-Pérez B, Reyes M, Rojas L, Ocaña B, Pérez B, Kosky RG, Angenon G (2012) Establishment of embryogenic cell suspension cultures and Agrobacterium-mediated transformation in banana cv.‘Dwarf Cavendish’(Musa AAA): effect of spermidine on transformation efficiency Plant Cell. Tissue Organ Culture (PCTOC) 111:79–90CrossRefGoogle Scholar
- Dhekney SA, Li ZT, Zimmerman TW, Gray DJ (2009) Factors influencing genetic transformation and plant regeneration of Vitis American. J Enol Viticult 60:285–292Google Scholar
- Gindro K, Spring J, Pezet R, Richter H, Viret O (2006) Histological and biochemical criteria for objective and early selection of grapevine cultivars resistant to Plasmopara viticola. Vitis 45:191–196Google Scholar
- He P (1999) Viticulture. China agriculture press, BeijingGoogle Scholar
- Ingram DS (1981) Physiology and biochemistry of host–parasite interaction. In: Spencer DM (ed) The downy mildews, pp 143–161Google Scholar
- Jain S, Kumar D, Jain M, Chaudhary P, Deswal R, Sarin NB (2012) Ectopic overexpression of a salt stress-induced pathogenesis-related class 10protein (PR10) gene from peanut (Arachis hypogaea L.) affords broad spectrum abiotic stress tolerance in transgenic tobacco Plant Cell. Tissue Organ Cult (PCTOC) 109:19–31CrossRefGoogle Scholar
- Lafon R, Bulit J (1981) Downy mildew of the vine. In: Spencer DM (ed) The downy mildews, pp 601–614Google Scholar
- Petri C, Alburquerque N, Pérez-Tornero O, Burgos L (2005) Auxin pulses and a synergistic interaction between polyamines and ethylene inhibitors improve adventitious regeneration from apricot leaves and Agrobacterium-mediated transformation of leaf tissues. Plant Cell Tissue Organ Cult 82:105–111CrossRefGoogle Scholar
- Torregrosa L, Iocco P, Thomas M (2002) Influence of Agrobacterium strain, culture medium, and cultivar on the transformation efficiency of Vitis vinifera L. American. J Enol Viticult 53:183–190Google Scholar
- Wan Y, Schwaninger H, He P, Wang Y (2007) Comparison of resistance to powdery mildew and downy mildew in Chinese wild grapes. Vitis 46:132–136Google Scholar
- Wang Y, He P (1997) Study on inheritance of leaves’ resistance to powdery mildew in Chinese native wild. Vitis 30:19–25Google Scholar
- Wang Y, Liu Y, He P, Chen J, Lamikanra O, Lu J (1995) Evaluation of foliar resistance to Uncinula necator in Chinese wild Vitis species. Vitis 34:159–164Google Scholar