Walk in growth cabinets

For this study 20 seeds for each of the cultivar Hong Ke Zi (cold tolerant) and BTx623 (cold sensitive) were planted in ragdoll set up under cold stress temperatures. The temperatures were continuous 14 °C for cold stress using Conviron walk-in growth cabinets. The seedlings were allowed to grow under 14 h light/10 h dark conditions. Leaf tissue samples for RNA extraction were obtained from five seedlings per replicate for Hong Ke Zi and BTx623. Three replicates were extracted separately using the TRIzol reagent and company recommended protocols (Life Technologies, Grand Island, NY). The total RNA samples from each replicate were quantified using Nano Drop and subsequently purified using the RNAeasy mini clean up kit (Qiagen, Valencia, CA). To evaluate the expression differences observed, qRT-PCR primers were designed for four selected genes in the expression module using primer3 (Additional file 7) and sorghum actin gene was used for normalization. Briefly, qRT-PCR was performed using copy-DNA libraries generated from the total RNA using Invitrogen cDNA synthesis kit (Invitrogen, Grand Island, NY). qRT-PCR was performed on the cDNA of the four samples with 3 biological and 3 technical replicates using SybrGreen on LightCycler 480.

Two sorghum germplasm, (BTx623 and HongKeZi), with contrasting response to cold stress under controlled and field conditions were used in the study. HongKeZi (PI567946, www.ars-grin.gov) is a cold tolerant line classified under the Kaoliang-nervosum working group identified by Franks et al., [7 (link)]. BTx623, a combine type inbred line classified under the kafir_zera-zera working group, is sensitive to cold stress under field conditions and served as the cultivar used in reference genome sequence (http://phytozome.jgi.doe.gov/) for sorghum.
For this study 20 seeds for each of the cultivar were planted in ragdoll set up under control and cold stress temperatures. The temperatures were continuous 14 °C for cold stress and 28 °C for control temperature using Conviron walk-in growth cabinets. Each temperature was represented by three replicates. The seedlings were allowed to grow under 14 h light/10 h dark conditions. The plants were grown in each temperature for 7 days. Each replicate was represented by five uniform seedlings (shoots and roots).