Rapid regeneration of stable transformants in cultures of potato by improving factors influencing Agrobacterium-mediated transformation

Abstract

An efficient and rapid Agrobacterium tumefaciens-mediated transformation protocol was developed to generate activation-tagged mutant lines with the aim of large-scale functional analysis of the potato genome. The explants were inoculated with an Agrobacterium strain harboring the binary plasmid pSKI074 containing four CaMV 35S enhancers in the T-DNA region which activates the downstream genes in the host plant after its integration. Various parameters investigated to increase transformation efficiency were the type and age of explant, cultivar, hormone combinations, preculture of explants, period of co-cultivation with bacteria and concentration of bacterial cultures used for transformation. Stem explants from 5 week old plantlets of cv. Bintje which had undergone phytohormone pretreatment for 4 days, inoculation with diluted bacterial concentration of OD600 = 0.2 containing acetosyringone followed by 2 days of co-cultivation and selection in media with IAA and trans-zeatin all helped in greatly improving the transformation efficiency. The total time required from infection to rooted shoots was 6-7 weeks. Initial evidence for stable integration and expression of the transgenes by PCR analysis showed that over 93% of the regenerated lines were transgenic and this was confirmed by Southern hybridization

Transgenic Potato (Solanumtuberosum) Expressing Two Antifungal Thionin Genes Confer Resistance To Fusarium Spp.

“…So we tried another infection time (5min) 3-days co-cultivation and some explants survive and then transferred for cultivation MS media. This agree with [17] who used 5-min infection into two potato cultivars from Bangladesh (cardinal and heera), [31], from Molecular Plant Pathology Laboratory in USA, who worked on two Serbian potato cultivars (Dragaevka and Jelica) and [32] To sum up, the difference in infection periods of explants with Agrobacterium could be due to firstly, used different cultivars which could be differ in their sensitivity to infection with bacteria. Secondly it may be due to use different explants for infection (node, internode or leaf segment), finally it could be due to different environmental conditions which completely different differ from the environmental conditions in Egypt.In this study, we selectednodal explants for transformation because [23] proved that the nodal explants are much more resistant during manipulation and more amenable to in vitro conditions.…”

Hairy Root Induction on Justicia gendarussa by Various Density of Agrobacterium rhizogenes strain LB 510

“…The research was carried out by Chakravarty & Pruski (2010) showed that OD 600 = 0.2 could improve significantly the average efficiency transformation of Agrobacterium rhizogenes on potato.…”

Production of Transgenic Cucumber Plantlets Containing Sequences From Watermelon Mosaic Virus-Ii Fused With GFP Gene

“…These data are in agreement with Yin et al (2005), they showed that the frequency of Agrobacte-rium-mediated transformation ranged from 0.8 to 10% and was influenced by bacteria cell density. Chakravarty and Wang-Pruski (2010) reported that from various parameters investigated to increase transformation efficiency was concentration of bacterial cultures which used for transformation. Different concentration of bacterial suspension (OD600 0.2-0.6) were used.…”