RESULTS

Computational results

Germination rates

Phenotype analysis

Promoters insertion into cloning vector

Computational results

The results obtained from the in silico analysis are presented in the Project Modeling section.

Arabidopsis thaliana germination assays

Germination rate

The germination rates are shown in Table 1. These values were calculated as the quotient of the germinated seeds and the total number of seeds per plate.

Table 1 - Germination rates calculated for the three types of plates: Murashige and Skoog (MS), MS with Dimethyl sulfoxide (DMSO) and MS supplemented with spectinabilin.
	Culture medium
	MS	MS + DMSO (µL)					MS + Spectinabilin (µg/mL)
	MS	10	20	100	200	400	0.05	0.1	0.5	1	2
Germination rate	40/40	40/40	38/40	40/40	38/40	39/40	39/40	38/40	38/40	39/40	39/40

In Figure 1, it is shown a plate layout with both germinated and non-germinated seeds. Here, it is possible to observe that germinated seeds are easily identified by its green dicotyledonous. Also, we can observe a thin root growing out of the seed involucre with a closer look.

Figure 1 - Plate of MS medium with 400 µL of DMSO. (Right) Approximately 40 seeds were inoculated evenly in which only one non-germinated seed highlighted in red was observed. (Left) Germinated seeds macroscopic identification of dicotyledonous and root.

Overall, both DMSO and spectinabilin are responsible for a slight decrease in the germination rate, although this effect is not sufficient to compromise the germination and growth viability of Arabidopsis thaliana.

Phenotype analysis

The phenotype analysis was performed through the observation of all seeds in a microscope. In Figure 2, the differences identified in roots of seeds inoculated in MS medium supplemented with DMSO or spectinabilin are depicted and compared with the MS medium.

Figure 2 - Microscopic analysis of possible phenotypes triggered by the three different culture mediums.

Most seed roots which germinated in MS medium exhibit a curly and thick appearance. However, in the medium supplemented with DMSO or spectinabilin two distinct root forms are observed: one similar to the ones described previously and another one characterized by a thin and long filament appearance. Since spectinabilin is diluted in DMSO, it is not possible to conclude if the modified phenotype is a response only to the presence of DMSO or if spectinabilin also contributes to this aspect.

Promoters insertion into cloning vector pSEVA234

Both promoter constructions were amplified by PCR. To confirm the success of the PCR, we run an agarose gel electrophoresis. As shown in Figure 3, a band of the expected size is visible for both inserts which confirms both inserts were amplified by PCR.

Figure 3: Agarose gel electrophoresis (1% agarose) of PCR amplified products using species-specific PCR primer sets. Lane 1: 1kB plus DNA size marker (Thermo); Lane 2: Promoter 1-GFP-terminator PacI; Lane 3: Promoter 1-GFP-terminator EcoRI; Lane 4: Promoter 2-GFP-terminator PacI; Lane 4: Promoter 2-GFP-terminator EcoRI;