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Title:   Author  item
                                            SINGH, ALKA - University Of Californiaitem
                                            KUMAR, PUSHPENDRA - University Of Californiaitem
                                            Jiang, Cai-Zhongitem
                                            REID, MICHAEL - University Of California
  Submitted to: Vegetos: An International Journal of Plant Research
                                    Publication Type: Peer Reviewed Journal
                                    Publication Acceptance Date: 9/16/2013
                                    Publication  Date: 5/5/2014
                                    Publication URL: http://10.5958/j.2229-4473.26.2s.137Citation: Singh, A., Kumar, P., Jiang, C., Reid, M.S. 2014. TRV Based Virus Induced Gene Silencing in Gladiolus (Gladiolus grandiflorus L.), A Monocotyledonous Ornamental Plant. Vegetos: An International Journal of Plant Research. Vol. 26 (Special) : 170-174 (2013) DOI:10.5958/j.2229-4473.26.2s.137.Interpretive Summary: Virus-induced gene silencing (VIGS) provides a useful method for gene function analysis that avoids the need for time consuming transformation and regeneration. The Tobacco rattle virus (TRV) vector has proved to be very useful for VIGS in diverse dicotyledonous plant taxa due to its broad host range. It has been shown to function effectively in a range of taxa, including tobacco, tomato, petunia, Californian Poppy, and Mirabilis jalapa. It induces only very mild symptoms, infects large areas of adjacent cells and silences expression of genes in vegetative and floral meristems. Although TRV mediated VIGS has proved a powerful tool for studying gene function in dicotyledonous plants, it has not yet been shown to be effective in monocotyledonous taxa.

VIGS vectors for monocotyledonous plants have been constructed from Barley stripe mosaic virus (BSMV) and Brome mosaic virus (BMV). These vectors have been used for gene function analysis in barley, wheat, rice, maize, but the empty vector of BSMV causes leaf mosaic symptoms difficult to differentiate from the phytoene desaturase (PDS) silencing photobleaching phenotype.

TRV is known to infect a wide range of monocotyledonous plants, including geophytes such as gladiolus. But there has been no report of the use of the TRV VIGS vector in such plants. Many factors influence the efficiency of VIGS in different plant species, including inoculum concentration, inoculation method and organ, growth stage of the inoculated plants, and growing conditions after inoculation. We therefore tested VIGS in gladiolus (Gladiolus grandiflora L) using a Tobacco Rattle Virus (TRV) vector containing a fragment of the gladiolus gene encoding phytoene desaturase (PDS). Gladiolus corms and plants were infiltrated with cultures of Agrobacterium tumefasciens transformed with the vector system. We tested the effect of different inoculum concentrations, different inoculation strategies and different growing temperatures after inoculation. Successful gene silencing was obtained by syringe inoculation of the sprouted corms) with Agrobacterium at an OD600 of 2 using syringe infiltration of the young bud, and growing the plants at 18°C after inoculation. White streaks characteristic of silencing PDS were first observed four weeks after inoculation and symptoms continued to develop up to eight weeks after inoculation. Silencing was not observed in plants grown at other temperatures, inoculated with lower concentrations of Agrobacterium, or inoculated by other means (corm, leaf, or vacuum infiltration). This is the first report on the potential use of TRV based VIGS in gladiolus and this optimized procedure of the TRV induced gene silencing should further facilitate for highly efficient functional analysis of genes.
                                Technical Abstract: Virus-induced gene silencing (VIGS) has not yet successfully been used as a tool for gene functional analysis in non-grass monocotyledonous geophytes. We therefore tested VIGS in gladiolus (Gladiolus grandiflora L) using a Tobacco Rattle Virus (TRV) vector containing a fragment of the gladiolus gene encoding phytoene desaturase (PDS). Gladiolus corms and plants were infiltrated with cultures of Agrobacterium tumefasciens transformed with the vector system. We tested the effect of different inoculum concentrations, different inoculation strategies and different growing temperatures after inoculation. Successful gene silencing was obtained by syringe inoculation of the sprouted corms) with Agrobacterium at an OD600 of 2 using syringe infiltration of the young bud, and growing the plants at 18°C after inoculation. White streaks characteristic of silencing PDS were first observed four weeks after inoculation and symptoms continued to develop up to eight weeks after inoculation. Silencing was not observed in plants grown at other temperatures, inoculated with lower concentrations of Agrobacterium, or inoculated by other means (corm, leaf, or vacuum infiltration). This is the first report on the potential use of TRV based VIGS in gladiolus and this optimised procedure of the TRV induced gene silencing should further facilitate for highly efficient functional analysis of genes.


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