**2. Materials and methods**

#### **2.1 Source of seeds**

Viable seeds of *H. vulgare* sv13 were obtained from Kaap Agri Bedryf Ltd. located in Malmsbury, Western Cape. The seeds used originated from the Swartland District of the Western Cape. The seeds were first weighed on a balance that measures in 100 g increments.

## **2.2 Experimental design and hydroponic setup**

The experiment was carried out in the plant tissue culture laboratory at the Cape Peninsula University of Technology's Bellville Campus. A 230 cm × 450 cm growing room was used to control light and temperature and determine the best growing conditions. Shelving units measuring 200 cm in height, 127 cm in length, and 40 cm in depth were installed in the growing room. The shelving unit had six shelves that were 37 cm apart and measured 120 cm × 40 cm. Two fluorescent light bulbs were installed on each shelf. For drainage, a corrugated fiberglass sheet was cut to the size of the shelf below and positioned at a 55-degree angle. The front, bottom end was fitted with a D-shaped gutter. This was used to collect the runoff from the fiberglass sheets. The run-off was then directed back to a sump via the gutter, resulting in an ebb and flow closed watering system. After cleaning and soaking the seeds, they were placed in perforated aluminum containers measuring 10 cm × 20 cm. The perforations were evenly spaced across the tray's bottom surface, with approximately 2 cm between each perforation. There was no need for a medium because the seeds germinated and formed a root mat that held the seedlings in place. The seed trays were then placed on the fiberglass sheeting, and each tray was fitted with an irrigation tube. Irrigation water was delivered to the seeds in their respective trays using a pump (HJ 1542 submersible), which delivered 622.5 mL/min to each tray for 2 min, for a total of 1245 mL. The pump was linked to a timer (MajorTech model MTD7), which controlled the amount of water delivered to each tray [29, 30]. Before the treated seeds were placed in the growing system, the entire setup, including the sump, Perspex shelves, and seed containers, was thoroughly cleaned and disinfected. The sump was filled with deionized water containing a 20% sodium hypochlorite solution, and the system was flushed to disinfect all surfaces [13].

The temperature of the room was kept at 23°C, as it was found that a temperature range of 20–30°C did not have a significant impact on growth [25, 31]. Two Samsung Smart InverterTM air conditioners were used to regulate the temperature. Fresh air was brought into the growing chamber through heap filters from outside the building. Lighting was provided with fluorescent tubes [32, 33]. The fluorescent bulbs used were Osram (L36/640) cool white fluorescent tubes with a light output of 5.96 kilo lux. The ExTech—Heavy Duty Digital Light Meter, model number HD 400, was used to measure the intensity of the light. A Panasonic TB178K timer control unit was used to set the lighting system to provide a photoperiod of 16 h day/8 h night [34, 35].



*Seed Soaking Times and Irrigation Frequencies Affected the Nutrient Quality and Growth… DOI: http://dx.doi.org/10.5772/intechopen.104503*

#### **2.3 Treatment preparation**

There were 25 treatments, each with 10 repetitions. Each treatment included a presoaking period followed by a post-soaking irrigation period (**Table 1**). Each repetition began with 100 g of viable seeds placed in a sterile plastic container containing 500 mL of distilled water containing a 20% solution of sodium hypochlorite (bleach) at room temperature [8, 14]. It was decided to test a range of seed soaking times, namely: 1, 3, 8, 16 and 24 h, which was compared against the control of 16 h. After the allotted soaking time, the seeds were washed in running, deionized water and placed in their respective growing trays without being exposed to darkness. Each tray was 10 cm × 20 cm in size. This ensured that the washed seeds had a depth of 1 cm. After that, the containers were placed in the hydroponic system to germinate (**Figures 1** and **2**). The seeds were allowed to germinate and grow into a forage mat for 8 days at 23°C under a photoperiod of 16-h day/8-h darkness.

Drip irrigation tubes were used to flood each seed tray with 1245 mL of water, with the excess running off through drainage holes in the seed container. The runoff was collected and channeled back into the sump of the hydroponic system for reuse. When necessary, the sump was refilled with distilled water mixed with a 20% bleach solution to ensure disinfection. The five previously mentioned treatments were subjected to five different irrigation intervals, each with 10 repetitions. Flood irrigation was used to fill each seed tray with water every 2; 4; 8; 10; and 12 h, with the control being a 2 hourly water interval [24, 36].

#### **2.4 Data collection**

Before removing the seedlings from their trays at the end of the 8-day growing cycle, a grid of 2 cm × 2 cm blocks was placed over the surface of the container, dividing the space into 50 blocks. This was used to determine the average leaf height per block by measuring the height of each leaf in the respective 2 cm × 2 cm block. The average leaf height of the sample plants for each block was then measured to determine the container's overall average leaf height. The longest leaf in each tray was

#### **Figure 1.**

*Photograph showing the hydroponic setup and irrigation supplied to each tray.*

#### **Figure 2.** *Photograph of barley seedlings at harvest (photo by R.A. Smith).*

also measured and recorded. Thereafter the seedling mat was removed from its tray and the depth of the root mat was recorded to determine whether the initial 1 cm of soaked seed had expanded over the 8-day growing period.

For nutrient analysis, the trays were removed from the experiment and all excess remaining surface water was allowed to drain away after the allotted growth period of 8 days. The seedlings in their respective trays (**Figures 1** and **2**) were then weighed using a Kern KB 360-3 N scale that measures up to 0.01 g to determine their fresh weight. The weight of the seedling mat was calculated by subtracting the weight of the container from this measurement. Once the fresh weight of the plant material was determined, the seedling mat was removed from its tray and placed in brown paper bags before being dried in an oven (Labtech LDO-150F) at 60–70°C for 36–48 h. The plant material was weighed again after it had completely dried to determine its dry weight. Using a Culatti Typ MFC CZ13 mill, the dried plant material was ground and sieved after being weighed. Following that, samples of dried plant material were sent to the Agrifood Technology Station for protein and nitrogen analysis [37].
