Effect Of Different Physical Wounding On Transformation Efficiency Of Agrobacterium Mediated In Lily ’Robina’

In this study, the regenerated small scales of lily ‘Robina’ were used as expalnts forAgrobacterium-mediated transformation and treated with sonication, vacuum infiltration andvortexing with quartz sand, respetively. Observing the wounded explants by scanningelectron microscope and operating the experiment of induction of bulblets to analyse effectsof wounding on induction of bulblets. The GUS histochemical assay was performed toestimate the transient expression of the GUS gene in the transformed explants and the stableexpression in the transformed plantlets. PCR analysis of GUS gene and HPT gene were testedfor stable transformation efficiency. The main results obtained are as follows:1. Scanning electron microscopy revealed micro-wounds on the surface of all treatedtissues while the surface of the non-treated tissue was smooth and intact. And sonicationtreatment to the explants obtained the smallest wound, while vortexing with quartz sand gotthe biggest. The duration of the wounding exposure times during treatments had an effect onwound size: the longer the exposure of explants to sonication, vacuum infiltration andvortexing with quartz sand, the bigger the wound size.2. It was found that the explants treated with3min sonication,15min vacuuminfiltration and1min vortexing with quartz sand all obtained100%induction rate of bulblets.Moreover, a longer exposure of the tissues to the wounding treatment was observed todecrease the induction rate of bulblets.3. For the histochemical GUS assay, most of the transformed bulb scale explants treatedwith sonication befor Agrobacterium infection was all found blue foci. Moreover, explantstreated with3min sonication and1min vortexing with quartz sand displayed a substantialnumber of blue foci on the explants surface.4. In the histochemical GUS assay of hygromycin-resistant leaf, scale and root, all ofthem from treatment with3min sonication showed the blue coloration indicative of GUSactivity, and the leaf from treatment with15min vacuum infiltration showed blue colorationonly on the leaf vein while there was no blue coloration from vortexing with quartz sandtreatment. Therefor, vortexing with quartz sand is not suitable to assist Agrobacteriummediated transformation for lily ‘Robina’. 5. The highest hygromycin-resistant frequency,48.85%and transformation efficiency,28.30%were all obtained from3min sonication treatment. Overall,3min sonicationtreatment of bulb scale explants is an effective protocol for obtaining the high transformationefficiency of lily ‘Robina’.