Agrobacterium LBA

Ramessur, Anusha Devi, Bothwell, John H., Maggs, Christine A., Gan, Sook Yee & Phang, Siew Moi, 2018, Agrobacterium-mediated gene delivery and transient expression in the red macroalga Chondrus crispus, Botanica Marina (Warsaw, Poland) 61 (5), pp. 499-510 : 502-503

Agrobacterium LBA 4404 cells successfully attached to Chondrus cells

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 Chondrus thallus surface at the wound sites. Whole colonies were observed in a fibrillar mesh ( Figure 2 ).

Protocol for eliminating Agrobacterium using cefotaxime

The results of testing cefotaxime concentrations of 500 and 800 mg l−1 confirmed that this cefotaxime-supplemented wash protocol ensured that residual Agrobacterium and other culturable bacteria were eliminated on the thalli within 6 days ( Table 1 ).

Agrobacterium successfully delivered pCAMBIA 1301 into Chondrus thalli

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 Agrobacterium LBA 4404 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 Agrobacterium LBA 4404 bearing a construct where the GUS gene 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).

Successful expression obtained with the Chondrus -specific expression cassette

Using the Agrobacterium transformation 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 GUS precipitate 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.

Figures

Figure 2: Intimate contact and adhesion of bacteria to wound sites of Chondrus thalli. 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.

Figure 3: Histochemical staining and low-power microscopic analysis of Chondrus thallus segments for transient expression of GUS 7 days after co-cultivation. (A) Pin-pricked thalli co-cultivated without bacteria, (B) pin-pricked thalli co-cultivated with untransformed LBA4404 bacteria, (C) unwounded and untransformed thalli, (D) pin-pricked thalli co-cultivated with LBA4404/pRI 910 (no GUS gene), (E) thalli transformed with LBA4404 (pCAMBIA 1301), scale bar: 1 cm.

Figure 4: Effect of cultivation medium, wounding method and acetosyringone on Agrobacterium transformation efficiency. The transformation efficiencies of Chondrus thalli segments co-cultivated with pCAMBIA 1301-transformed Agrobacterium in seawater and Induction Medium (IM) were compared. Using seawater as co-cultivation medium, the wound-related treatments, namely biolistics wounding, wounding using pin-pricks and no wounding, were compared. The effect of acetosyringone (100 µM) was also studied. Data are expressed as means ± SE (n = 4). The Mann-Whitney non-parametric test was conducted to compare between two groups.

Figure 5: GUS expression in Chondrus thallus segments transformed with LBA 4404/pRI 910 (Ac-GUS). (A) Surface view of control thallus not co-cultivated with Agrobacterium, (B) surface view of transformed thallus showing GUS expression at wound sites, (C) section through untransformed thallus, (D) thallus section within wounded area showing blue medullary cells at wound spots, (E) cortical cells from untransformed control thallus, (F) GUS-expressing cortical cells in transformed thallus, (G) medullary filaments from untransformed control thallus, (H) medullary filaments from transformed thallus.