In vitro Conditions for Dark Growth and Analysis of Maize Seedlings

Background

Soilless media and temperature-controlled environments have been used for over eighty years to generate reproducible plant growth (McClary, 1940; George et al., 2008). Murashige and Skoog (MS) media, containing salts, amino acids and vitamins, is one of the most commonly used plant growth medias (Murashige and Skoog, 1962). MS was originally developed for tobacco but has since been used to grow other plant species including Arabidopsis and maize. This growth media does not readily react with phytohormones unlike other soilless media such as peat, perlite and vermiculite (Best et al., 2014 and 2017; Malik et al., 2014). Therefore, hormones can be added to the growth media to compare responses between wild-type and mutant growth.

The rolled paper towel method is the standard for high throughput quantitative analysis of maize seedling germination (Phaneendranath, 1980). Maize kernels are sterilized, and rolled in moist paper towels, then allowed to grow. Because microbes can influence plant growth, this method often includes a fungicide solution in addition to kernel surface sterilization (Pace et al., 2014). Rolled or vertical filter paper can also be used to grow maize seedlings in combination with hormone application (Camp and Wickliff, 1981; Hahn et al., 2008). Other methods for growing maize in soilless environments may use custom-built systems (Singletary and Below, 1989).

Plants grown in the absence of light undergo skotomorphogenesis (Josse and Halliday, 2008). This developmental program causes the stem and leaves of plants to elongate via anisotropic cell expansion toward potential light sources (Cheng et al., 2000; Markelz et al., 2003; Wang et al., 2016). Dark growth can be achieved by light-controlled growth chambers (Wang et al., 2016), or by placing maize seedlings grown in clear plastic boxes with moist cellulose filter paper into light-tight black boxes (Waller and Nick, 1997). Dark-growth of Arabidopsis has been achieved by covering plates in aluminum foil (Alabadí et al., 2004).

We combined a number of these popular methods to create a controlled growth environment for dark-grown maize seedlings. We first sterilized maize kernels from the B73 inbred background (Martínez and Wang, 2009). Next, kernels were grown in soil or on solid MS media in clear plastic (“magenta”) boxes and subjected to either standard (16-h light, 8-h dark) or constant dark conditions using aluminum foil to block light (Alabadí et al., 2004). These boxes are commercially available, relatively inexpensive, maintain sterile conditions, can withstand repeated autoclave cycles, and are easy to cover with foil to block light. However, their use is limited to maize seedlings less than two-weeks old due to their relatively small size. We found that the B73 mesocotyls grown in soil were longer in both standard and dark treatments compared to standard and dark treated seedlings grown on ½ MS media solidified with agar. However, we observed that the mesocotyl lengths of B73 seedlings grown on ½ MS were more consistent. Taken together, we show that the method described here creates a reproducible environment for quantitative analysis of dark-grown maize mesocotyl length. This method could also be used to study maize seedling growth responses to hormones, abiotic stress conditions, or other treatments.