Successful floral‐dipping transformation of post‐anthesis lisianthus (Eustoma grandiflorum) flowers

Abstract

The adaptation of the Agrobacterium-mediated floral-dipping technique is limited, to date, to a small number of plants. In this paper, we present the efficient transformation of one of the leading plants in the cut flower industry, lisianthus (Eustoma grandiflorum). This method is approximately 18 months shorter than the known tissue culture-based transformation. The Excalibur Pink cultivar and two additional breeding lines, X-1042 and X-2541, were transformed using three different marker genes (benzyl alcohol acetyltransferase (BEAT) originating from Clarkia breweri, the feedback-insensitive bacterial gene AroG*, and the empty pART27 vector expressing a kanamycin-resistance cassette (nptII)). Genomic transformation was successful in all tested cases with transformation efficiency ranked from 0.2 to 2.9%, which is well in the range of results from Arabidopsis studies. Unlike Arabidopsis, in which floral-dipping transformation was efficient only at a pre-anthesis stage before ovary sealing, lisianthus flowers were transformed when dipping occurred 4 days pre-anthesis or 3-5 days post-anthesis with 1.5 and 3.7% efficiencies, respectively. Post-anthesis transformation occurred when the flower ovaries were sealed. Flower dipping of Excalibur Pink flowers with fluorescent Agrobacterium containing a GFP marker gene demonstrated Agrobacterium entrance into the sealed flower ovary through the open stigma and style tube. In this study, we demonstrated floral-dipping transformation of a commercial plant, lisianthus Excalibur Pink, occurring after sealing of the ovaries, probably via the stigma and wide open style tunnel.

Agrobacterium-mediated vacuum infiltration and floral dip transformation of rapid-cycling Brassica rapa

“…However, neither of them could get transformants from crossing when inoculated plants served as pollen donors and non-inoculated plants served as pollen recipients [21, 23]. Recently, it was reported that prairie gentian ( Eustoma grandifloru ; also called lisianthus) was successfully transformed via the floral dip method at a post-anthesis stage when ovaries were partially sealed, while in these flowers a stylar channel remained open [18]. Another study recently reported tomato transformation by floral bud injection [17], another approach to providing Agrobacterium with access to ovules.…”

“…Compared to tissue culture, such methods are typically less time-consuming and labor-intensive [15]. Confirmed successes with in planta transformation had previously been limited to plants in the Brassicaceae, but successes have now been reported for Eustoma grandiflorum , Setaria viridis and Solanum lycopersicum [16–18]. Pakchoi ( Brassica rapa ssp.…”

Increased substrate availability reveals the potential of scentless lisianthus flowers in producing fragrant benzenoid‐phenylpropanoids

“…Plants were hand pollinated and the seeds were harvested after 2 months. Seedlings were transferred to selection media containing 50 mg/L kanamycin 1 month after germination (Fang et al, 2018).…”

“…The lisianthus cultivar "Excalibur Pink" was chosen for transformations due to its vigor and the recent efficient and relatively fast flower-dipping transformation method developed for this cultivar (Fang et al, 2018). "Excalibur Pink" plants were transformed with both AroG* and C. breweri BEAT constructs (Figure 2(A)) and screened as described in Fang et al (2018). The putative transgenic plants were then analyzed for genomic integration, expression of the transgene, and for AroG* protein accumulation (Figure 2(B)-(D)).…”

“…In this study, to overcome the apparent substrate availability limitation and reveal the full potential of lisianthus plants to produce fragrant benzenoid‐phenylpropanoids, we transformed the commercial lisianthus cultivar “Excalibur Pink” with both AroG* and BEAT genes under constitutive promoters. For faster and more efficient production of transgenic lisianthus, the recently developed Agrobacterium ‐mediated floral‐dipping transformation methodology was used (Fang et al, 2018). The results presented here clarify that lisianthus flowers have the potential for production of several fragrant BPVs given high substrate availability: All the transgenic lines accumulated methyl benzoate, a fragrant BPV downstream the biosynthetic pathway, not detected in WT plants.…”