無菌状態で小さな分子に対する植物の反応を評価するための柔軟な低コスト化の養液栽培システム

A simple, versatile, and low cost in vitro hydroponics system was successfully optimized, enabling large scale experiments under sterile conditions. This system facilitates application of chemicals in solution and their efficient absorption by roots for molecular, biochemical, and physiological studies. We demonstrated that this system was highly efficient in obtaining homogeneous seedlings along initial development, as well as separating roots and shoots using arabidopsis thaliana seeds.

The following items are necessary to build up the hydroponics system and cultivate plants under sterile, controlled conditions. 7%ethanol, 10%bleach solution, polysorbate 20, MS medium including vitamins. MES monohydrate, agar, potassium hydroxide.

Laminar flow hood, hot plate, growth chamber. Disposable plastic boxes, sterile glass Petri dishes, adhesive tape, sterile 200 and 300 microliters pipet tips, scissors, 300 microliters multichannel pipet, 200 microliters pipet, scalpel blade, tweezers, and 200 microliters pipet tip racks. It's crucial that the pipet tip flat has an area to be filled with the solid medium.

Sterilize the pipet tips racks without covers that will be used as mini-tanks in the autoclave at 121 degrees for 20 minutes. In order to avoid contamination, all of the previously mentioned materials were sterilized in the autoclave. When this was not possible, as in the case of the disposable plastic boxes, the materials were cleaned with 7%ethanol before entering the laminar flow hood.

If the hood allows, UV light can be turned on for 10 minutes prior to the assembling of the hydroponic system. Seal the upper surface of the pipet tip flat with the adhesive tape. If possible, let them under UV light for 10 minutes.

Then, add 108 microliters of melted solid medium in each well using a multichannel pipet. If you are preparing many tanks, use a hot plate to prevent the MS medium from solidifying. Allow the medium to solidify completely, about 30 minutes.

During the solidification period, UV light can be turned on. Fill up the tip box completely with liquid culture medium. Guarantee the close compact between solid and liquid media.

Remove adhesive tapes off the upper surface of the pipet tip flat, and fit it on the rack carefully. The hydroponic system is now ready to receive the sterilized seeds. The liquid bleach sterilization described here is a practical method to sterilize seeds.

Fist, place 500 arabidopsis seeds in a 1.5 ml micro tube. Use as many micro tubes as necessary according to the number of plants required for the experiment. Wash the seeds with 7%ethanol for two minutes with agitation.

Remove the ethanol carefully. Add one ml of 10%bleach solution containing two microliters of polysorbate 20 detergent. Agitate for five minutes.

Remove the solution carefully. Finally, rinse the seeds with sterilized distilled water until all the bleach residue is completely removed, approximately five times. This protocol is feasible for arabidopsis, however, this system can also be used for other plant species.

To this end, said sterilization procedure should be modified according to the species requirement. After surface sterilization, seeds were immersed in sterile distilled water and stratified at four degrees in the dark for five days to synchronize germination. Cut slightly the extremity of 200 microliters pipet tip with the aid of a sterile scalpel.

Pipet the arabidopsis seeds into the solid culture medium on the upper surface of the pipet tip flat. Take care for the medium not to loosen from the flat, otherwise the seeds will be shade and the seedlings will not grow properly. Store as many mini-tanks as possible inside a disposable plastic box to maintain a high humidity and the environment free from microorganisms.

Seal the disposable plastic box using adhesive tape. The hydroponic systems are now ready for entering the growth chamber. This hydroponic system was initially developed to facilitate the administration of chemicals to plants, such as isotope labeled compounds, which in general are very expensive to be applied in large scale experiments.

As a proof of concept, we used the ATP competitive inhibitor AZD8055, which specifically targets the ATP binding site of the target rapamycin protein kinase. TOR kinase is a major regulator integrating nutrient sensing and energy signaling to promote cell proliferation and growth. In order to assess primary responses of TOR inhibition mediated by AZD, seeds of arabidopsis were grown hydroponically until desired developmental stage under 12 hour photo period.

Fresh medium containing two micromolar AZD or 0.05%DMSO as control were replaced in the hydroponic tanks at the end of the night. Seedlings were harvested at different time points after treatment, separated into roots and shoots frozen in liquid nitrogen, ground into a fine powder, and stored at minus 80 degrees until use. The phosphorylation pattern of ribosomal protein S6 kinase, RPS6, one of the well-known target of the TOR pathway.

Immunoblotting shows the amount of total and phosphorylated RPS6 in roots, graph A, and shoots, graph B.Repression of phosphorylation in the presence of ADZ855 occurred as soon as 30 minutes after drug treatment in both roots and shoots, demonstrating that under the experimental conditions used, ADZ has also shown to be a potent TOR inhibitor which rapidly represses its kinase activity. Transgenic arabidopsis lines with reduced expression of the TOR gene or components of the TOR complex present a clear starch excess phenotype. Qualitative analysis of starch using Lugol's solution reveal the expected pattern of starch accumulation and degradation during the cycle.

Seedling that did not receive application of the MSO or AZD855 showed no greater accumulation of starch in their leaves at the end of the night, and starch accumulation in control plants, the MSO, was consistent with the literature. Furthermore, plants treated with AZD presented greater amount of remaining starch at the end of the night when compared to the control seedlings. These results indicate the usefulness of the proposed hydroponic system in growing seedlings mimicking physiological conditions.

Starch accumulates in leaves during the day, and is remobilized over night to sustain metabolic activities. Under normal conditions, only a small fraction of starch, between 5 and 10%of the total amount after the end of the day remains at the end of the night. These results are tested that starches phenotype observed under TOR repression occurs all over the cycle.

Hydroponically grown plants were compared to seedlings growing horticultural substrate under very similar climactic conditions concerning the expression level of the abscisic acid have responsive element binding factor three, ABF3 gene, shown in figure 5-A. The expression of ABF3 directly correlates with internal ABA levels, a class of hormones widely known as a marker due to its role in multiple abiotic stress responses. Although hydroponically grown plants did present a significant increase in ABF3 expression when compared to plants grown in soil, the expression levels of asparagine synthetase one ASN1, were not affected by the MSO or AZD treatments shown in figure 5-B.

However, the expression of trehalose phosphate synthase five, TPS5, plus significant increase after eight hours of TOR repression. ASN1 and TPS5 respond to low and high sugar levels respectively, suggesting that these plants were not experienced in genetic stress. We have succeeding using this system to evaluate the application of small molecules in plants.

In summary, this hydroponic system possesses many advantages because it's very fast and easy to assembly, and has a low cost, as the major components are cheap, and can be extensively reused. Furthermore, this system is versatile, enabling the study of intact seedlings or distinctions along plant development, and it's highly scalable, allowing the cultivation of a huge number of plants in a very small area.