Protocol: Optimizing Hydroponic Growth Systems For Nutritional and Physiological Analysis Of Arabidopsis Thaliana and Other Plants
Hydroponic growth systems are a convenient platform for studying whole plant physiology. However, we found through trialing systems as they are described in the literature that our experiments were frequently confounded by factors that affected plant growth, including algal contamination and hypoxia. We also found the way in which the plants were grown made them poorly amenable to a number of common physiological assays.
- The drivers for the development of this hydroponic system were:
- the exclusion of light from the growth solution;
- to simplify the handling of individual plants, and
- the growth of the plant to allow easy implementation of multiple assays. These aims were all met by the use of pierced lids of black microcentrifuge tubes. Seed was germinated on a lid filled with an agar-containing germination media immersed in the same solution.
Following germination, the liquid growth media was exchanged with the experimental solution, and after 14-21 days seedlings were transferred to larger tanks with aerated solution where they remained until experimentation. We provide details of the protocol including composition of the basal growth solution, and separate solutions with altered calcium, magnesium, potassium or sodium supply whilst maintaining the activity of the majority of other ions. We demonstrate the adaptability of this system for: gas exchange measurement on single leaves and whole plants; qRT-PCR to probe the transcriptional response of roots or shoots to altered nutrient composition in the growth solution (we demonstrate this using high and low calcium supply); producing highly competent mesophyll protoplasts; and, accelerating the screening of Arabidopsis transformants. This system is also ideal for manipulating plants for micro pipette techniques such as electrophysiology or SiCSA.
We present an optimized plant hydroponic culture system that can be quickly and cheaply constructed, and produces plants with similar growth kinetics to soil-grown plants, but with the advantage of being a versatile platform for a myriad of physiological and molecular biological measurements on all plant tissues at all developmental stages. We present ‘tips and tricks’ for the easy adoption of this hydroponic culture system.
Arabidopsis thaliana (L.) Heynh. (Arabidopsis) has been adopted as a model plant of choice in many laboratories for a variety of reasons. These include a brief life cycle, a small and well-annotated genome, its amenability to tissue culture, the limited cell-layers per cell type (for developing roots), the availability of natural diversity sets and targeted mutants, and the ease at which it can be genetically transformed. The diminutive stature and rosette growth habit of Arabidopsis also means that it does not require a large area to cultivate. At the same time, the size of Arabidopsis has presented considerable challenges for those wanting to perform physiological measurements on intact plants such as gas exchange, hydraulic conductance, or for obtaining single-cell parameters such as turgor pressure and membrane potential. To benefit from the vast molecular resources of Arabidopsis, physiologists have had to adapt measuring equipment and assays to the microscale; these technological challenges have curtailed the use of Arabidopsis as a tractable physiological model. In order to perform such assays whilst providing a flexible experimental platform for manipulation of both the shoot and root environment, the use of hydroponics for research purposes has become common.
Hydroponics, as a convenient means for studying plants in the laboratory and for growing commercial crops, was a term first coined by William F. Gericke in 1929, yet it is a documented technique dating back to the late 17th century. Its advantages include the potential for accessibility to all plant tissues and the easy manipulation of the nutrient profile of the growth medium when compared to soil, given the complex interaction of ions with soil particles. Agar or phytagel plates share these advantages but the opportunities for physiological experimentation using this system are limited as seedlings can only be grown for about 2 weeks on plates and plants transpire very little meaning that sucrose is commonly included as a carbon substrate and aseptic culture must be used. A disadvantage of both hydroponics and agar plates is that many species have a different root morphology when compared to soil, including a lack of root hairs, although this is not the case for Arabidopsis. Various hydroponic systems have been developed for the growth of Arabidopsis independently in several laboratories reflecting their need and utility; Araponics©, is an example of a commercially available system. Other hydroponics systems described in the literature have often been designed with a specific purpose in mind, and as a result have not been tested in terms of the ease at which various experimental parameters can be Whilst trialling these methods in our laboratory we identified several key limitations with these systems as they are documented in the literature, including:
the use of a small holding tank (up to 1 L) to hold the growth solution, reducing scalability;
the need to sterilise parts of the set-up, which lengthened and complicated the procedure;
the use of rockwool or sponge, which prevented access to the full root system and predisposed the apical meristems to flooding;
the use of specialised materials such as a prefabricated seed holder, which increased cost ; and the need to transfer plantlets between multiple growth environments. While each methodology possessed strengths and was designed to suit its endpoint analysis, we sought to streamline the entire process and provide a universal and fully adaptable system.
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- Amir Ali Shaikh