Agrobacterium tumefaciens-mediated transformation of Lotus tenuis and regeneration of transgenic lines

Abstract

A protocol for the production of transgenic plants was developed for Lotus tenuis via Agrobacteriummediated transformation of leaf segments. The explants were co-cultivated (for 3 days) with an A. tumefaciens strain harbouring either the binary vector pBi RD29A:oat arginine decarboxylase (ADC) or pBi RD29A:glucuronidase (GUS), which carries the neomycin phosphotransferase II (nptII) gene in the T-DNA region. Following co-cultivation, the explants were cultured in Murashige and Skoog medium supplemented with naphthalenacetic acid (NAA) and benzyladenine (BA) and containing kanamycin (30 lg ml -1 ) and cefotaxime (400 lg ml -1 ) for 45 days. The explants were subcultured several times (at 2-week intervals) to maintain the selection pressure during the entire period. About 40% of the explants inoculated with the pBiRD29:ADC strain produced eight to ten adventitious shoots per responsive explant through a direct system of regeneration, whereas 69% of the explants inoculated with the pBi RD29A:GUS strain produced 13-15 adventitious shoots per responsive explant. The selected transgenic lines were identified by PCR and Southern blot analysis. Three ADC transgenic lines were obtained from 30 infected explants, whereas 29 GUS transgenic lines were obtained from 160 explants, corresponding to a transformation efficiency of 10 and 18.1%, respectively.More than 90% of the in vitro plantlets were successfully transferred to the soil. The increase in the activity of arginine decarboxylase from stressed ADC-Lt19 lines was accompanied by a significant rise in the putrescine level. The GUS transgenic line driven by the RD29A promoter showed strong signals of osmotic stress in the leaves and stem tissues. All of the transgenic plants obtained exhibited the same phenotype as the untransformed controls under non-stress conditions, and the stability of the gene introduced into the cloned materials was established.

Activity of the Arabidopsis RD29A and RD29B promoter elements in soybean under water stress

“…When implementing genetic construct designs with these promoter elements in heterologous systems as a means to regulate expression of targeted transgenes with the objective to improve abiotic stress response in plants, it is imperative to gain an understanding on whether the fidelity of regulation from their native context translates to the plant system under consideration. In Lotus tenuis, for example, introduction of a RD29A-GUS cassette revealed induction of GUS expression upon exposure to a simulated abiotic stress, but only in leaves and stems, not roots (Espasandin et al 2010).…”

Long-term preservation of Lotus tenuis adventitious buds

“…The cryopreservation experiments were carried out with adventitious bud clusters (ABCs) obtained following the regeneration protocol previously reported (Espasandin et al 2010). Leaf segments excised from leaves of the first to fourth nodes of 30-day-old in vitro seedlings of L. tenuis cv.…”

“…Many agronomics and organoleptic traits distinguish the different ecotype which is using in the breeding program to produce seeds for monoculture pastures. In this context, we previously developed a protocol for mass propagation and transformation of L. tenuis (Espasandin et al 2010) to improve the tolerance to drought (Espasandin et al 2014) and salinity (Espasandin et al 2017). The possibility to rescue wild ecotypes, propagate selected genotypes, generate transgenic plants for the incorporation of pest, diseases and stress tolerance traits, creates the need to have a procedure that allows the conservation of genetic resources.…”

Expression of CoQ10-producing ddsA transgene by efficient Agrobacterium-mediated transformation in Panicum meyerianum

“…However, the transformation efficiency for these two species was not very high. High efficiency of Agrobacterium-mediated transformation is relevant to the high regeneration ability of plants from tissue culture (Espasandin et al 2010;Li et al 2010;Zhang et al 2010); however, warm-season grasses generally have low tissue culturability (Glowacka et al 2010;Panaia et al 2009). We previously investigated efficient tissue culture systems for Panicum spp., and found that Panicum meyerianum Nees has high tissue culturability (Seo et al 2008(Seo et al , 2010.…”