Development of Efficient Plant Regeneration and Transformation System for Impatiens Using Agrobacterium tumefaciens and Multiple Bud Cultures as Explants

Abstract

BackgroundImpatiens (Impatiens walleriana) is a top selling floriculture crop. The potential for genetic transformation of Impatiens to introduce novel flower colors or virus resistance has been limited by its general recalcitrance to tissue culture and transformation manipulations. We have established a regeneration and transformation system for Impatiens that provides new alternatives to genetic improvement of this crop.ResultsIn a first step towards the development of transgenic INSV-resistant Impatiens, we developed an efficient plant regeneration system using hypocotyl segments containing cotyledonary nodes as explants. With this regeneration system, 80% of explants produced an average of 32.3 elongated shoots per initial explant plated, with up to 167 elongated shoots produced per explant. Rooting efficiency was high, and 100% of shoots produced roots within 12 days under optimal conditions, allowing plant regeneration within approximately 8 weeks. Using this regeneration system, we developed an efficient Agrobacterium-mediated Impatiens transformation method using in vitro multiple bud cultures as explants and a binary plasmid (pHB2892) bearing gfp and nptII genes. Transgenic Impatiens plants, with a frequency up to 58.9%, were obtained within 12 to 16 weeks from inoculation to transfer of transgenic plants to soil. Transgenic plants were confirmed by Southern blot, phenotypic assays and T1 segregation analysis. Transgene expression was observed in leaves, stems, roots, flowers, and fruit. The transgenic plants were fertile and phenotypically normal.ConclusionWe report the development of a simple and efficient Agrobacterium-mediated transformation system for Impatiens. To the best of our knowledge, there have been no reports of Agrobacterium-mediated transformation of Impatiens with experimental evidence of stable integration of T-DNA and of Agrobacterium-mediated transformation method for plants using in vitro maintained multiple bud cultures as explants. This transformation system has the advantages of 1) efficient, simple and rapid regeneration and transformation (with no need for sterilization or a greenhouse to grow stock plants), 2) flexibility (available all the time) for in vitro manipulation, 3) uniform and desirable green tissue explants for both nuclear and plastid transformation using Agrobacterium-mediated and biolistics methods, 4) no somaclonal variation and 5) resolution of necrosis of Agrobacterium-inoculated tissues.

A Perspective on Hypericum perforatum Genetic Transformation

“…However, it should be recalled that use of these compounds individually did not help in H. perforatum transformation in our previous attempts (Franklin et al, 2007 ). Nevertheless, application of a mixture of antioxidants could be useful, as it has been shown to improve the efficiency of Agrobacterium -mediated transformation in a number of other recalcitrant plant species (Dan, 2008 ; Dan et al, 2010 , 2015 ). It is also important to understand the signaling events that trigger H. perforatum defense activation upon Agrobacterium interaction.…”

Genetic Stability in Melon Transformation System: Assessment by Flow Cytometry and ISSR Markers

“…Ntui et al (2010) reduced ethylene production by explants after Agrobacterium infection, even as the ACC gene expression reduced and increased the transformation rate. A cytological study by Dan et al (2010) on melon epidermal and sub-epidermal cells revealed a lower regeneration rate of the infected explants which can be related to the disability of cells in causing regeneration. Also, this may be due to the genetic instability of the infected meristematic parts.…”

Robustness and evolvability in the B-system of flower development

“…), the petaloid sepals of Impatiens (Geuten et al, 2006) or the first whorl tepals from Tricyrtis (Liliales). These species have indeed been reported to be stably transformable (Adachi et al, 2005;Nakano et al, 2007;Feng et al, 2009;Dan et al, 2010).…”