Agrobacterium-mediated gene delivery and transient expression in the red macroalga Chondrus crispus Ramessur, Anusha Devi Bothwell, John H. Maggs, Christine A. Gan, Sook Yee Phang, Siew Moi Botanica Marina 2018 Warsaw, Poland 2018-09-12 61 5 499 510 QZ9 LBA LBA [824,1095,1673,1703] Alphaproteobacteria Rhizobiaceae Agrobacterium Bacteria Rhizobiales 3 502 Proteobacteria genus  Under the SEM, short, rod-shaped bacterial cells about 0.8 × 2 µm were seen attached in large numbers (an estimated average of one bacterial cell per µm 2) to the  Chondrusthallus surface at the wound sites. Whole colonies were observed in a fibrillar mesh ( Figure 2).   Figure 2: Intimate contact and adhesion of bacteria to wound sites of  Chondrusthalli. Thalli co-cultivated for 48 h in induction medium were fixed and viewed under a scanning electron microscope: (A) control thallus incubated without bacteria showing a clean bacterium-free surface; (B) close up view of rod-shaped bacteria attached to thallus surface at wound site; (C) bacterial film at wound sites on thallus surface.  Protocol for eliminating  Agrobacteriumusing cefotaxime The results of testing cefotaxime concentrations of 500 and 800 mg l−1confirmed that this cefotaxime-supplemented wash protocol ensured that residual  Agrobacteriumand other culturable bacteria were eliminated on the thalli within 6 days ( Table 1).     Agrobacteriumsuccessfully delivered pCAMBIA 1301 into  Chondrusthalli The methodology for transformation developed using pCAMBIA 1301 followed by the established wash protocol included controls to detect any false positives occurring from indigenous activity of beta-glucoronidase in  Chondrus(unwounded and untransformed thalli, Figure 3C), indigenous activity from LBA4404 bacterial cells (pin-pricked thalli co-cultivated with untransformed  AgrobacteriumLBA4404 bacteria, Figure 3B), physiological response of algal cells to wounding (pin-pricked thalli cultivated without bacteria, Figure 3A) and vector components (pin-pricked thalli co-cultivated with  AgrobacteriumLBA4404 bearing a construct where the  GUSgene is absent; pRI 910 binary vector, Figure 3D). None of these controls were stained. However, thalli transformed with pCAMBIA 1301 were stained blue ( Figure 3E).  Manipulating transformation conditions can enhance transformation efficiency As shown in Figure 4, among the pin-pricked thalli, a significant increase in transformation efficiency (higher percentage of thallus segments stained blue) was observed when seawater was used in preference to Induction Medium (Mann-Whitney test, p = 0.029) in the presence of acetosyringone. However, no transformation was detected in the unwounded thallus segments or those wounded by bombardment when co-cultivation was conducted using seawater. In addition, without the presence of acetosyringone, transformation occurred at a significantly lower efficiency of less than 10% (Mann-Whitney, p = 0.004).   Table 1: Confirmation of the suitability of 500 and 800 mg l−1 cefotaxime in eliminating bacteria.     Mean number of colonies (per plate)   Day  Concentration of cefotaxime (mg l − 1 )   0  500  800   0 > 1000 > 1000 > 1000   2 > 300  0.75 ± 0.95  1.25 ± 0.95   4 > 300  0.25 ± 1.60  2.00 ± 0.50   6 > 300  0  1.00 ± 1.2   8 >300 0 0     Agrobacteriumco-cultured with thalli was eliminated by consecutive washes and incubated in fresh antibiotic. Every 2 days, the thallus segments were rinsed for 4 min with sterile seawater six times before incubation for 48 h in fresh seawater with replenished cefotaxime. A thallus segment was streaked on a LBA plate which was incubated at 28° C for up to 4 days to check for the persistence of  Agrobacterium. This was conducted in triplicates (n= 3). Colonies appearing from periodic streaks of thalli on LBA plates were counted.  Successful expression obtained with the  Chondrus-specific expression cassette Using the  Agrobacteriumtransformation protocol developed using pCAMBIA 1301, the constructed pRI 910 (Ac- GUS) was delivered into thallus cells using seawater as the co-cultivation medium and with the same controls as for pCAMBIA 1301. Blue colouration was apparent at the sites of tear, cut or wounding on  Agrobacterium-mediated transformed thalli when compared to controls ( Figure 5). Transverse sections through the tissue revealed blue localization of the GUSprecipitate within the peripheral cortical cells and central medullary filaments. In general, a darker staining intensity was observed with pRI 910 (Ac- GUS). Furthermore, a higher transformation efficiency of 85% was obtained with this  Chondrus-specific construct as opposed to 52% with pCAMBIA 1301.